Anti-psma adc conjugate compositions and methods of use thereof

Abstract

The present invention relates to prostate-specific membrane antigen antibodies (anti-PSMA) and anti-PSMA antibody drug conjugates (ADCs). The present invention provides a method for treating a subject with an anti-PSMA ADC to inhibit, prevent, or treat a PSMA-related disease or cancer, including prostate cancer. The present disclosure also provides pharmaceutical compositions containing an anti-PSMA ADC suitable for administration to a human subject.

Description

[0001] Cross-referencing This application claims the benefit of U.S. Provisional Application No. 63 / 578,131, filed August 22, 2023; U.S. Provisional Application No. 63 / 589,823, filed October 12, 2023; U.S. Provisional Application No. 63 / 591,090, filed October 17, 2023; and U.S. Provisional Application No. 63 / 591,665, filed October 19, 2023, the entire contents of each of which are incorporated herein by reference. Technical Field

[0002] This invention discloses novel anti-prostate-specific membrane antigen (anti-PSMA) antibodies and antibody-drug conjugates. More specifically, this invention discloses the use of anti-PSMA antibody-drug conjugates in inhibiting, preventing, or treating PSMA-related diseases or cancers. This invention also discloses pharmaceutical formulations containing anti-PSMA antibody-drug conjugates. Background Technology

[0003] Prostate cancer is the most frequently diagnosed non-skin-related malignancy among men in developed countries. It is estimated that one in six men will be diagnosed with prostate cancer. Diagnosis of prostate cancer has improved with the use of serum-based biomarkers such as prostate-specific antigen (PSA). Furthermore, prostate tumor-associated antigens have provided targets for tumor imaging, diagnosis, and targeted therapy. Prostate-specific membrane antigen (PSMA), a prostate tumor-associated marker, is one such target. PSMA is significantly overexpressed in androgen-independent prostate cancer. PSMA overexpression is associated with a high tumor grade, a high risk of disease progression, and recurrence (Perner S. et al., Human Pathology, 2007, 38(5):696-701). High PSMA expression is associated with poor clinical prognosis and significantly shorter survival.

[0004] High levels of PSMA expression have been found in prostate cancer, and specifically in metastatic castration-refractory prostate cancer (mCRPC). PSMA has also been found in a variety of other solid tumors. Prostate cancer represents a significant unmet need. In 2018, there were 1,300,000 new cases of prostate cancer worldwide, with a five-year survival rate of approximately 27% and 359,000 associated deaths. Prostate cancer is the second leading cause of death among men in the United States (Siegel RL et al., CA: A Cancer Journal for Clinicians; 2023, 73:17–48). Treatment options include surgery, radiation therapy, and androgen deprivation therapy (ADT); however, patients with advanced prostate cancer eventually progress to mCRPC, with a median overall survival of approximately 2 years (Khoshkar Y. et al., BJUI Compass, 2022, 3:173–83). Although approved treatments for mCRPC patients include different classes of drugs, such as taxanes, androgen receptor pathway inhibitors, pembrolizumab for high microsatellite instability (MSI-) or mismatch repair deficient subgroups, radiotherapy, and PARP inhibitors for patients with BRCA mutations (Lowrance W. et al., Journal of Urology, 2023, 209:1082–90), patients eventually develop resistance to these treatments, and the disease remains incurable.

[0005] PSMA is a promising therapeutic target for prostate cancer, with limited expression in healthy cells and high overexpression rates on the surface of primary prostate tumors as well as in metastatic lesions in lymph nodes and bone (Hupe MC et al., Front. Oncol, 2018, 8:623; Queisser A. et al., Modern Pathology, Elsevier, 2015, 28:138–45). In 2022, based on results from the Phase III VISION trial, the radiotherapy Pluvicto was approved for mCRPC (Sartor O. et al., N Engl J Med. 2021, 385:1091–103), clinically validating PSMA as a therapeutic target for prostate cancer. PSMA is also internalized, making it an ideal target for antibody-drug conjugates (ADCs) that deliver cytotoxic drugs to tumor cells via binding to target antigens on tumor cells, internalization, and subsequent release of the cytotoxic payload (i.e., the free payload) into the cytoplasm (Liu H. et al., Rajasekaran AK, Moy P, Xia Y, Kim S, Navarro V et al., Cancer Res. 1998, 58:4055–60). Multiple PSMA-targeting ADCs using different payloads and adapter technologies have been previously developed and evaluated in clinical trials (Petrylak DP et al., Prostate, 2020, 80:99–108; Petrylak DP et al., Prostate, 2019, 79:604–13; Milowsky MI et al., Urol. Oncol., 2016, 34:530, e15-530.e21; de Bono JS et al., Clin Cancer Res. 2021, 27:3602–9). However, these ADCs appear to have discontinued their clinical development, likely due to unacceptable toxicity and / or a narrow therapeutic index.

[0006] There is a need for improved treatments targeting PSMA and PSMA-related cancers. To overcome this deficiency in the art, this disclosure provides anti-PSMA ADCs and pharmaceutical compositions containing them, as well as methods of treating human subjects with PSMA-expressing diseases or cancers using said ADCs and compositions. Summary of the Invention

[0007] This invention discloses anti-PSMA antibody pharmaceutical conjugates for the treatment of PSMA-related diseases or cancers. In some embodiments, this disclosure provides anti-PSMA antibody pharmaceutical conjugates for inhibiting, preventing, or treating PSMA-related conditions, disorders, diseases, or cancers. This invention also provides stable pharmaceutical formulations containing anti-PSMA antibody pharmaceutical conjugates suitable for administration to human subjects.

[0008] In some general aspects, this disclosure provides a method for treating cancer, the method comprising: Administer an effective amount of an anti-PSMA antibody-drug conjugate (ADC) to a human subject in need, wherein the anti-PSMA ADC comprises: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0009] In some embodiments, each heavy chain includes the heavy chain variable region of SEQ ID NO: 1. In some embodiments, each light chain includes the light chain variable region of SEQ ID NO: 2. In some embodiments, each heavy chain includes the heavy chain variable region of SEQ ID NO: 1, and each light chain includes the light chain variable region of SEQ ID NO: 2.

[0010] In some embodiments, each heavy chain amino acid sequence is SEQ ID NO: 8, which contains pAF incorporated at Kabat position 114 (i.e., amino acid position 116 in SEQ ID NO: 8). In some embodiments, each light chain amino acid sequence is SEQ ID NO: 9. In some embodiments, each heavy chain amino acid sequence is SEQ ID NO: 8, which contains pAF incorporated at Kabat position 114 (i.e., amino acid position 116 in SEQ ID NO: 8), and each light chain amino acid sequence is SEQ ID NO: 9. In some embodiments, the anti-PSMA ADC is ARX517.

[0011] In some embodiments, the effective amount of the anti-PSMA ADC is at least about 1.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is up to about 5 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least about 1.4 mg / kg and up to about 5 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least about 1.4 mg / kg and up to about 3.4 mg / kg of human subject body weight.

[0012] In some embodiments, the effective amount of the anti-PSMA ADC is about 1.4 mg / kg, about 1.7 mg / kg, about 2 mg / kg, about 2.4 mg / kg, about 2.9 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 4.3 mg / kg, about 4.5 mg / kg, about 4.7 mg / kg, or about 5 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 1.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 1.7 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 2 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is greater than 2.0 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 2.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is greater than 2.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least 2.5 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 2.9 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is greater than 2.9 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least 3.0 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.1 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.2 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.3 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.5 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.6 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.7 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.8 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 3.9 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 4.0 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 4.3 mg / kg of human subject body weight.In some embodiments, the effective amount of the anti-PSMA ADC is about 4.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 4.5 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 4.7 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is about 5 mg / kg of human subject body weight.

[0013] In some embodiments, a method of treating cancer includes administering an effective amount of an anti-PSMA antibody-drug conjugate (ADC) to a human subject in need, wherein the effective amount of the anti-PSMA ADC is greater than 2.0 mg / kg and at most about 5.0 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is greater than 2.0 mg / kg and at most about 3.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least about 2.4 mg / kg and at most about 3.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least about 2.8 mg / kg and at most about 3.4 mg / kg of human subject body weight. In some embodiments, the effective amount of the anti-PSMA ADC is at least about 3.0 mg / kg and at most about 3.4 mg / kg of human subject body weight.

[0014] In some embodiments, an effective amount of the anti-PSMA ADC is administered to human subjects according to a dosing schedule. In some embodiments, the dosing schedule is once every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the dosing schedule is once every two weeks. In some embodiments, the dosing schedule is once every three weeks. In some embodiments, the dosing schedule is more than once within a three-week cycle.

[0015] In some embodiments, the effective dose of the anti-PSMA ADC is about 1.4 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 1.7 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 2.0 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 2.4 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 2.9 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 3.0 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 3.1 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 3.2 mg / kg human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 3.3 mg / kg of human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 3.4 mg / kg of human subject body weight, administered every three weeks. In some embodiments, the effective dose of the anti-PSMA ADC is about 3.5 mg / kg of human subject body weight, administered every three weeks.

[0016] In some implementations, the human subject has prostate cancer or non-prostate cancer that expresses PSMA. In some implementations, the PSMA-expressing cancer is a low-PSMA-expressing cancer. In some implementations, the PSMA-expressing cancer is a moderately PSMA-expressing cancer. In some implementations, the PSMA-expressing cancer is a high-PSMA-expressing cancer.

[0017] In some embodiments, the human subject has prostate cancer. In some embodiments, the prostate cancer is metastatic castration-refractory prostate cancer (mCRPC). In some embodiments, the human subject has previously received taxane therapy. In some embodiments, the prostate cancer has progressed after prior taxane therapy. In some embodiments, the cancer is hormone-refractory prostate cancer. In some embodiments, the cancer is resistant or refractory to prior standard therapies used to treat prostate cancer. In some embodiments, the human subject has previously been treated with abiraterone, dalolutamide, apalutamide, or enzalutamide.

[0018] In some embodiments, the method of treating with the anti-PSMA ADC provided herein delays or inhibits cancer progression in human subjects. In some embodiments, the method increases the survival of human subjects compared to the median survival of subjects who have not previously received the same or different anti-PSMA ADCs. In some embodiments, the method increases the survival of human subjects, wherein the survival of human subjects is increased compared to the median survival of subjects with taxane-resistant cancer expressing anti-PSMA who have not previously received the same or different anti-PSMA ADCs.

[0019] In some embodiments, the method of treatment with the anti-PSMA ADC provided herein reduces the circulating level of CTCs in human subjects compared to baseline levels of CTCs. In some embodiments, the method reduces or stabilizes serum PSA levels in human subjects compared to baseline levels of prostate-specific antigen (PSA). In some embodiments, the method reduces serum PSA levels in human subjects compared to baseline levels of PSA. In some embodiments, the reduction in serum PSA levels in human subjects is at least about 30% compared to baseline levels of PSA. In some embodiments, the reduction in serum PSA levels in human subjects is at least about 50% compared to baseline levels of PSA. In some embodiments, the reduction in serum PSA levels in human subjects is at least about 90% compared to baseline levels of PSA.

[0020] In some implementations, the anti-PSMA ADC is administered intravenously.

[0021] In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC described herein provides a serum terminal half-life of at least about 5 days for the anti-PSMA ADC. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment provides a serum terminal half-life of the anti-PSMA ADC in the range of about 5 days to about 10 days.

[0022] In some implementations, following administration of an effective amount of the anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC described herein provides a time (Tmax) of at least approximately 5 days for the free payload to reach its maximum serum concentration, wherein the free payload released from the anti-PSMA ADC is pAF-AS269 having the following structure (see...).Figure 8C ): ; Or its salts. In some embodiments, the treatment provides at least about 6 days of free payload Tmax after administration of an effective amount of anti-PSMA ADC to a human subject. In some embodiments, the treatment provides about one week of free payload Tmax after administration of an effective amount of anti-PSMA ADC to a human subject.

[0023] In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC described herein provides a maximum free payload serum concentration (Cmax) of up to about 1 ng / mL. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment method provides a free payload Cmax of up to about 0.5 ng / mL, up to about 0.4 ng / mL, up to about 0.3 ng / mL, or up to about 0.2 ng / mL. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment method provides a free payload Cmax in the range of about 0.01 ng / mL to about 0.3 ng / mL.

[0024] In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC provided herein provides a maximum serum concentration (Cmax) of at least about 20 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL of anti-PSMA ADC. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC provided herein provides a maximum serum concentration (Cmax) of at least about 20 μg / mL of anti-PSMA ADC. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC provided herein provides a maximum serum concentration (Cmax) of at least about 30 μg / mL of anti-PSMA ADC. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of treatment with the anti-PSMA ADC provided herein provides a maximum serum concentration (Cmax) of at least about 40 μg / mL of anti-PSMA ADC. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of anti-PSMA ADC treatment described herein provides a serum maximum concentration (Cmax) of at least about 50 µg / mL of anti-PSMA ADC. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the method of anti-PSMA ADC treatment described herein provides a serum maximum concentration (Cmax) of at least about 60 µg / mL of anti-PSMA ADC.

[0025] In some implementations, the method of treatment with the anti-PSMA ADC described herein provides at least about 50% reduction in circulating tumor DNA (ctDNA) after administration of an effective amount of anti-PSMA ADC to human subjects.

[0026] In some aspects, methods of treating a subject with cancer using the anti-PSMA ADC of this disclosure further include administering an effective amount of another therapeutic agent. In some embodiments, the therapeutic agent is a chemotherapeutic agent, a hormonal agent, an antitumor agent, an immunostimulant, an immunomodulatory agent, or an immunotherapeutic agent, or a combination thereof. In some embodiments, the therapeutic agent is a hormonal agent. In some embodiments, the hormonal agent is enzalutamide. In some embodiments, the therapeutic agent is a checkpoint inhibitor, a PSMA kinase inhibitor, a cyclin-dependent kinase inhibitor, a tyrosine kinase inhibitor, a small molecule kinase inhibitor, or a platinum-based therapeutic agent.

[0027] In some other general aspects, this disclosure provides a pharmaceutical composition comprising an effective amount of an anti-PSMA antibody-drug conjugate (ADC), wherein the anti-PSMA ADC comprises: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain contains the heavy chain variable region of SEQ ID NO:1, and one non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, and each light chain contains the light chain variable region of SEQ ID NO:2; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0028] In some embodiments, this disclosure provides a pharmaceutical composition comprising an effective amount of an anti-PSMA antibody-drug conjugate (ADC), wherein the anti-PSMA ADC comprises: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein the amino acid sequence of each heavy chain is SEQ ID NO:8, wherein one non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, and the amino acid sequence of each light chain is SEQ ID NO:9; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0029] In some embodiments, the pharmaceutical composition comprises an anti-PSMA ADC, namely ARX517.

[0030] In some embodiments, the pharmaceutical composition comprises: (i) an anti-PSMA ADC at a concentration ranging from about 5 mg / mL to about 25 mg / mL; and (ii) a pharmaceutically acceptable component selected from the group consisting of sucrose, histidine buffer, and polysorbate 80, and combinations thereof; wherein the composition pH is in the range of about 5.5 to about 6.5. In some embodiments, the pharmaceutical composition comprises an anti-PSMA ADC at a concentration of about 10 mg / mL. In some embodiments, the sucrose concentration is in the range of about 5% (w / v) to about 15% (w / v); the histidine buffer concentration is in the range of about 15 mM to about 25 mM; and the polysorbate 80 concentration is in the range of about 0.001% (w / v) to about 0.02% (w / v). In some embodiments, the pharmaceutically acceptable component consists essentially of sucrose, histidine buffer, and polysorbate 80. In some embodiments, the composition pH is in the range of about 5.6 to about 6.2. In some embodiments, the composition comprises essentially the following: an anti-PSMA ADC at a concentration of about 10 mg / mL; sucrose at a concentration of about 9% (w / v); a histidine buffer at a concentration of about 20 mM; and polysorbate 80 at a concentration of about 0.01% (w / v); wherein the composition has a pH of about 5.9 ± 0.3. In some embodiments, the histidine buffer comprises essentially L-histidine, L-histidine hydrochloride, and water.

[0031] In some embodiments, the pharmaceutical composition comprises: (i) ARX517 at a concentration ranging from about 5 mg / mL to about 25 mg / mL; and (ii) a pharmaceutically acceptable component selected from the group consisting of sucrose, histidine buffer, and polysorbate 80, and combinations thereof; wherein the composition has a pH ranging from about 5.5 to about 6.5. In some embodiments, the pharmaceutical composition comprises ARX517 at a concentration of about 10 mg / mL. In some embodiments, the sucrose concentration ranges from about 5% (w / v) to about 15% (w / v); the histidine buffer concentration ranges from about 15 mM to about 25 mM; and the polysorbate 80 concentration ranges from about 0.001% (w / v) to about 0.02% (w / v). In some embodiments, the pharmaceutically acceptable component consists essentially of sucrose, histidine buffer, and polysorbate 80. In some embodiments, the composition has a pH ranging from about 5.6 to about 6.2. In some embodiments, the composition comprises essentially the following: ARX517 at a concentration of about 10 mg / mL; sucrose at a concentration of about 9% (w / v); histidine buffer at a concentration of about 20 mM; and polysorbate 80 at a concentration of about 0.01% (w / v); wherein the composition has a pH of about 5.9 ± 0.3. In some embodiments, the histidine buffer comprises essentially L-histidine, L-histidine hydrochloride, and water.

[0032] In some embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, the composition is stored frozen. In some embodiments, the composition is stored frozen at about -20°C. In some embodiments, the composition is stored refrigerated. In some embodiments, the composition is stored frozen at about 5°C ± 3°C.

[0033] In some embodiments, the anti-PSMA ADC of the pharmaceutical compositions provided herein comprises a charged variant. In some embodiments, the charged variant comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance. In some embodiments, the anti-PSMA ADC main substance has an isoelectric point (pI) of about 8.3. In some embodiments, the anti-PSMA ADC acidic substance has a pI of about 8.1. In some embodiments, the anti-PSMA ADC basic substance has a pI of about 8.4.

[0034] In some embodiments, the anti-PSMA ADC main substance is present in an amount of about 40% to about 70%. In some embodiments, the anti-PSMA ADC acidic substance is present in an amount of about 20% to about 40%. In some embodiments, the anti-PSMA ADC basic substance is present in an amount of about 5% to about 30%. In some embodiments, the sum of the percentages of each main substance, acidic substance, and basic substance is 100%.

[0035] In some embodiments, the percentages of the major substance, acidic substance, and basic substance are based on the UV area percentage at 214 nm of the integral of the corresponding major substance elution peak, acidic substance elution peak, and basic substance elution peak in the cation exchange chromatogram. In some embodiments, the percentages of the major substance, acidic substance, and basic substance are determined by cation exchange chromatography substantially as described in Example 17, wherein the cation exchange chromatogram is obtained by cation exchange chromatography. In some embodiments, the cation exchange chromatogram is compared with Figure 21A and... Figure 21B Consistent.

[0036] In some embodiments, the pharmaceutical composition has a drug-to-antibody ratio (DAR) in the range of about 1.5 to about 2.5. In some embodiments, the pharmaceutical composition has a drug-to-antibody ratio (DAR) in the range of about 1.9 to about 2.1.

[0037] In some aspects, this disclosure provides pharmaceutical compositions containing anti-PSMA ADCs for use in methods of treating cancer in human subjects of need.

[0038] It should be understood that the methods and compositions described herein are not limited to the specific methods, protocols, cell lines, constructs, and reagents described herein, and therefore can be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which is defined only by the appended claims. Attached Figure Description

[0039] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of exemplary embodiments, which utilize the principles of the invention and are illustrated in the accompanying drawings.

[0040] Figure 1A to Figure 1DPeptide maps of (A) unconjugated ARX517 mAb and ARX517 ADC showing the amberstatin 269 (AS269) conjugation site are depicted; (B) HIC chromatograms of unconjugated ARX517 mAb, DAR1, and DAR2 substances are shown; (C) SEC quantification of ARX517 ADC size variants are shown; and (D) thermal transition temperatures of unconjugated ARX517 and ARX517 ADC are determined by DSC.

[0041] Figure 2A to Figure 2E In vitro dose-response curves of ARX517 ADC and monomethylaurestatin E (MMAE) in various prostate cancer cell lines were plotted. (A) 22Rv1 (B) C4-2 (C) LNCaP (D) MDA-PCa-2b (E) PC3.

[0042] Figure 3A to Figure 3C Sensing traces from biolayer interference binding assays of humanized J591 mAb (ARX517 mAb) bound to (A) human PSMA, (B) cynomolgus monkey PSMA and (C) rat PSMA were depicted.

[0043] Figure 4A to Figure 4B The pharmacokinetics (PK) of ARX517 after administration to (A) tumor-free mice and (B) mice with C4-2 tumors were depicted. Serum concentrations of total antibody (TA) and ARX517 ADC (intact ADC) over time were also described.

[0044] Figure 5 Tumor growth inhibition (TGI) curves were plotted for mice carrying MDA-PCa-2b tumors. TGI% is shown in parentheses.

[0045] Figure 6A to Figure 6B Tumor growth inhibition in mouse prostate cancer models was depicted: (A) after administration of ARX517 ± enzalutamide in the TM00298 PDX model; and (B) after administration of ARX517 or enzalutamide in the CTG-2440 PDX model. TGI% is shown in parentheses.

[0046] Figure 7A to Figure 7B Tumor growth inhibition in an enzalutamide-resistant C4-2 CDX mouse model was depicted following administration of (A) a single dose of ARX517; and (B) repeated doses of ARX517 ± enzalutamide. TGI% is shown in parentheses.

[0047] Figure 8A to Figure 8CThe pharmacokinetics (PK) of ARX517 after administration to cynomolgus monkeys were depicted: (A) serum concentrations of total antibody (TA) and ARX517 ADC over time; (B) serum concentrations of pAF-AS269 over time; and (C) the structure of pAF-AS269, showing the drug-linker AS269 conjugated to pAF via an oxime bond.

[0048] Figure 9 The pK data depicted for the highest non-severe toxic dose of ARX517 in monkey toxicology studies relative to the pharmacologically active dose of ARX517 in mice carrying C4-2 tumors demonstrate the clear therapeutic index of ARX517.

[0049] Figure 10 The structure of ARX517 ADC was depicted, in which AS269 is conjugated to a pAF residue via a stable oxime bond, which is incorporated into each heavy chain of huJ591 mAb at position 114 (Kabat number).

[0050] Figure 11 The amino acid sequences of the ARX517 mAb heavy chain (HC) (SEQ ID NO:8) and light chain (LC) (SEQ ID NO:9) are depicted, respectively. Complementarity-determining regions are underlined. “U” indicates a non-naturally encoded amino acid (pAF) site that is genetically encoded and biosynthetically incorporated into amino acid position 114 (Kabat number) of each heavy chain.

[0051] Figure 12 The design of the first human dose escalation and dose expansion study for ARX517 was described.

[0052] Figure 13A to Figure 13B Low serum concentrations of free payload observed at all doses were depicted, with a free payload to ADC molar ratio of 0.06%. (A) Patients administered 2 mg / kg Q3W; (B) Patients administered the specified dose Q3W.

[0053] Figure 14A to Figure 14F The nearly overlapping TA and ADC PK curves were plotted, indicating strong stability at all dose levels. (A) 0.64 mg / kg; (B) 1.07 mg / kg; (C) 1.4 mg / kg; (D) 1.7 mg / kg; (E) 2 mg / kg; and (F) 2.4 mg / kg.

[0054] Figure 15 The half-life of ARX517 at a specified dose level Q3W was depicted. ARX517 exhibits a long half-life of approximately 6 to 10 days at doses ≥1.4 mg / kg.

[0055] Figure 16A to Figure 16B The increase in drug exposure proportional to the ARX517 dose is depicted. (A) ARX517 exposure AUC; (B) ARC517 Cmax.

[0056] Figure 17 The percentage change in PSA from baseline was depicted for cohorts 1 through 8 as the ARX517 dose increased.

[0057] Figure 18 The percentage change in PSA from baseline was depicted in cohorts 6 through 8. At the putative treatment dose (≥2.0 mg kg), 52% (12 / 23) of patients in cohorts 6 through 8 experienced a ≥50% decrease in PSA.

[0058] Figure 19 The percentage change in ctDNA levels from baseline was depicted in cohorts 4 through 8. A reduction of ≥50% in circulating tumor DNA (ctDNA) was observed in 81% (17 / 21) of patients (cohorts 4 through 8).

[0059] Figure 20 The percentage change from baseline based on RECIST v1.1 criteria was depicted in cohorts 4 through 8. A reduction in target lesions was observed in 56% (5 / 9) of patients (cohorts 4 through 8). PD = progressive disease; SD = stable disease; cPR = confirmed partial response.

[0060] Figure 21A to Figure 21B Representative chromatograms (A) and their developed views (B) obtained from cation exchange high-performance liquid chromatography of ARX517 solution for intravenous infusion are depicted; elution peaks corresponding to the main substance ARX517 and its acidic and basic charge variants are shown. Detailed Implementation

[0061] Before describing the invention in detail, it should be understood that the invention is not limited to specific methods, compositions, or biological systems, which can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, unless expressly stated otherwise, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, etc.

[0062] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Various modifications, alterations, and alternatives will be contemplated by those skilled in the art without departing from the invention. It should be understood that various alternative forms of the embodiments of the invention described herein may be used in the practice of the invention. The following claims are intended to define the scope of the invention and to cover methods and results falling within the scope of these claims and their equivalents.

[0063] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references.

[0064] The term “about” should be understood by those skilled in the art and may vary to some extent depending on the context in which it is used.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Various methods, materials, etc., similar to or equivalent to those described herein may be used in the practice or testing of the invention described herein.

[0066] All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the chemistry, chemical synthesis, compositions and other methods described in the publications, and may be used in conjunction with the invention currently described. The publications provided for discussion herein are provided only because their publications predate the filing date of this application.

[0067] The term "amino acid" refers to naturally occurring and non-natural or non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrolysine and selenocysteine. Amino acid analogs are compounds having the same basic chemical structure as naturally occurring amino acids, and by way of example only, having an α-carbon bound to a hydrogen, carboxyl, amino, and functional R group. Such analogs may have modified R groups (e.g., ortholeucine) or may have modified peptide backbones while still retaining the same basic chemical structure as naturally occurring amino acids. Non-limiting examples of amino acid analogs include homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acids are represented in this paper by their names, their commonly known three-letter symbols, or the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Nucleotides are represented by their generally accepted single-letter codes.

[0068] "Amino or carboxyl-terminal modifying groups" refers to any molecule that can be attached to a terminal amine group or a terminal carboxyl group, respectively. As an example, such terminal amine or carboxyl groups may be located at the ends of polymeric molecules, including but not limited to peptides, polynucleotides, and polysaccharides. Terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides, or proteins. As an example only, terminal modifying groups include polyethylene glycol or serum albumin. Terminal modifying groups can be used to modify the therapeutic properties of polymeric molecules, including but not limited to increasing the serum half-life of peptides, polypeptides, or proteins.

[0069] The term "antibody" as used herein refers to a protein composed of one or more polypeptides, substantially encoded by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, genes for the constant regions of κ, λ, α, γ (IgG1, IgG2, IgG3, and IgG4), δ, ε, and μ, as well as numerous genes for the variable regions of immunoglobulins. The antibodies described herein are also intended to include full-length antibodies and antibody fragments, and include antibodies naturally present in any organism, antibody variants, engineered antibodies, and antibody fragments. The antibodies described herein are also intended to include complete antibodies, monoclonal antibodies, or polyclonal antibodies. The antibodies described herein also encompass multispecific antibodies and / or bispecific antibodies. The antibodies of this invention include human antibodies. Human antibodies typically consist of two light chains and two heavy chains, each comprising a variable region and a constant region. The variable region of the light chain contains three CDRs, identified herein as CDRL1, CDRL2, and CDRL3 flanked by frame regions. The variable region of the heavy chain contains three CDRs, identified herein as CDRH1, CDRH2, and CDRH3 flanked by frame regions.

[0070] The term "antibody fragment" in this document refers to any form of antibody other than the full-length form. Antibody fragments in this document include antibodies as smaller components present within a full-length antibody, as well as engineered antibodies, such as antibody variants. Antibody fragments include, but are not limited to, Fv, Fc, Fab and (Fab')2, single-chain Fv (scFv), bivalent antibodies, trivalent antibodies, tetravalent antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains and variable regions, as well as alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard & Georgiou, Annu. Rev. Biomed. Eng. 2:339-76, 2000; Hudson, Curr. Opin. Biotechnol. 9:395-402, 1998). Another functional substructure is the single-chain Fv (scFv), which consists of variable regions of immunoglobulin heavy and light chains covalently linked by peptide linkers (Hu et al., Cancer Research, 56, 3055-3061, 1996). These small (Mr 25,000) proteins typically retain antigen specificity and affinity within a single polypeptide and can provide convenient structural units for larger antigen-specific molecules. Unless otherwise specified, statements and claims using the term "antibody" specifically include "antibody fragments".

[0071] As used herein, the term "antibody-drug conjugate" or "ADC" refers to an antibody molecule or fragment thereof covalently bonded to one or more bioactive molecules. The bioactive molecule can be conjugated to the antibody via a linker, polymer, or other covalent bond. ADCs are a class of efficient therapeutic constructs that allow for the targeted delivery of cytotoxic agents to target cells, such as cancer cells. Due to their targeting function, these compounds exhibit a much higher therapeutic index than the same systemically delivered drug. ADCs have been developed as whole antibodies or antibody fragments, such as scFv. The antibody or fragment is linked to one or more copies of a drug (e.g., the toxic moiety) via a linker that is stable under physiological conditions but can be cleaved once inside the target cells.

[0072] As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the complete antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragments, which are composed of V... L V H C L and C H1 (ii) A monovalent segment composed of structural domains; (iii) A divalent segment consisting of two Fab segments connected by disulfide bonds at the hinge region; (iv) A segment composed of V H and C H1 (iv) Fd fragments composed of structural domains; V-shaped segments formed by the antibody single arm L and V H The Fv segment, composed of structural domains, (v) is composed of V H dAb fragments composed of structural domains (Ward et al., Nature 341:544-546, 1989); (vi) separated complementarity-determining regions (CDRs), such as V with or without additional sequences (connectors, framework regions, etc.). H CDR3 and (v) are combinations of two to six separate CDRs, with or without additional sequences (joints, frame regions, etc.). Furthermore, although the two structural domains V of the Fv segment... L and V H Encoded by individual genes, but they can be linked together using recombination methods via synthetic adapters that allow them to be made into single polypeptide chains, where V L and V HRegions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, Bird et al., Science 242:423-426, 1988); and (Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibody. Furthermore, antigen-binding fragments include binding domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide), (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The hinge region can be modified by substituting one or more cysteine ​​residues with serine residues to prevent dimerization. Such binding domain immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments that function in the same manner as the intact antibody are screened.

[0073] Typical antigen-binding sites consist of variable regions formed by the pairing of light chain and heavy chain immunoglobulins. Antibody variable regions are structurally very homogeneous and exhibit highly similar structures. These variable regions typically consist of relatively homologous frame regions (FRs) spaced apart by three hypervariable regions called complementarity-determining regions (CDRs). The overall binding activity of the antigen-binding fragment is generally determined by the sequence of the CDRs. FRs typically play a role in the proper positioning and alignment of the CDRs in all three dimensions to achieve optimal antigen binding. In fact, because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies exhibiting the properties of specific naturally occurring antibodies by constructing expression vectors comprising CDR sequences from specific naturally occurring antibodies grafted onto frame sequences of different antibodies with different properties (see, for example, Riechmann, L. et al., Nature 332:323-327, 1998; Jones, P. et al., Nature 321:522-525, 1986; and Queen, C. et al., Proc.Natl.Acad.USA 86:10029-10033, 1989). Such frame sequences are available from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include the fully assembled variable genes formed during B cell maturation via V(D)J linkage. The germline gene sequence will also differ from that of the high-affinity secondary library antibody, which contains mutations throughout the variable gene but typically clusters in the CDR. For example, somatic mutations are relatively uncommon in the N-terminal portion of frame region 1 and the C-terminal portion of frame region 4. Furthermore, many somatic mutations do not significantly alter the antibody's binding properties. Therefore, it is not necessary to obtain the complete DNA sequence of a specific antibody to reconstruct a complete recombinant antibody with binding properties similar to the original antibody. Partial heavy and light chain sequences spanning the CDR region are usually sufficient for this purpose. These partial sequences are used to determine which germline variable gene segments and linker gene segments contribute to the recombinant antibody variable gene. The germline sequence is then used to fill in the missing portions of the variable region. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add the missing sequences, the cloned cDNA sequence can be combined with synthetic oligonucleotides via ligation or PCR amplification. Alternatively, the entire variable region can be synthesized to produce a fully synthetic variable region clone. This method offers certain advantages, such as eliminating or including specific restriction sites, or optimizing specific codons. Of course, all or part of the frame region of the antibody described herein can be used in conjunction with a CDR to optimize antibody affinity, specificity, or any other desired properties.

[0074] As used herein, "ARX517" refers to an anti-PSMA antibody-drug conjugate (ADC) comprising a humanized anti-PSMA monoclonal antibody, the humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:8, wherein a non-natural amino acid, para-acetyl-L-phenylalanine (pAF), is incorporated into each said heavy chain at position 114 according to Kabat numbering, and each light chain having the amino acid sequence of SEQ ID NO:9; wherein one drug-linker is conjugated to each said pAF via an oxime bond, and each said drug-linker is amberstatin269 (AS269) having the following structure: .

[0075] The structure of ARX517 is shown in Figure 10 , and SEQ ID NOs:8 and 9 are shown in Figure 11 and Table 3, which shows SEQ ID NOs:8 and 9 related to Variant 1 and the pAF incorporation site at Kabat amino acid position 114 (i.e., amino acid position 116 in SEQ ID NO:8).

[0076] In some embodiments, the present invention relates to polymers, such as bifunctional polymers. A “bifunctional polymer,” also known as a “bifunctional connector,” refers to a polymer comprising two functional groups capable of reacting specifically with other moieties to form covalent or non-covalent bonds. Such moieties may include, but are not limited to, natural or non-natural amino acids or side groups on peptides containing such natural or non-natural amino acids. Other moieties that may be attached to a bifunctional connector or bifunctional polymer may be the same or different moieties. By way of example only, a bifunctional connector may have a functional group that reacts with a group on a first peptide and another functional group that reacts with a group on a second peptide, thereby forming a conjugate comprising a first peptide, a bifunctional connector, and a second peptide. Many methods and connector molecules for attaching various compounds to peptides are known. See, for example, European Patent Application 0188256; U.S. Patent Nos. 4,659,839; 4,414,148; 4,699,784; 4,680,338; and 4,569,789, the entire contents of which are incorporated herein by reference. A "multifunctional polymer," also known as a "multifunctional linker," is a polymer containing two or more functional groups capable of reacting with other moieties. Such moieties may include, but are not limited to, natural or non-natural amino acids or side groups on peptides containing such natural or non-natural amino acids (including, but not limited to, amino acid side groups) to form covalent or non-covalent bonds. Bifunctional or multifunctional polymers can be of any desired length or molecular weight and can be selected to provide a specific desired spacing or conformation between one or more molecules linked to the compound and molecules bound thereto or bound to the compound.

[0077] As used herein, the term "bioavailability" refers to the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in systemic circulation. An increase in bioavailability refers to an increase in the rate and extent to which a substance or its active portion is delivered from a pharmaceutical dosage form and becomes available at the site of action or in systemic circulation. As an example, an increase in bioavailability can be expressed as an increase in the concentration of the substance or its active portion in the blood compared to other substances or active portions.

[0078] The terms “bioactive molecule,” “bioactive moiety,” or “bioactive agent,” as used herein, mean any substance that can affect any physical or biochemical property of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. Specifically, as used herein, a bioactive molecule includes, but is not limited to, any substance intended for the diagnosis, cure, relief, treatment, or prevention of disease in humans or other animals, or for otherwise enhancing the physical or mental health of humans or animals. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. The types of bioactive agents suitable for use with the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, contrast agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroidal and nonsteroidal drugs, and microbial-derived toxins.

[0079] "Modulation of bioactivity" refers to increasing or decreasing the reactivity of a peptide, altering its selectivity, or enhancing or decreasing its substrate selectivity. Analysis of the bioactivity of modified peptides can be performed by comparing the bioactivity of non-natural peptides with that of natural peptides.

[0080] In some embodiments, this disclosure relates to amino acids that have been incorporated into antibodies via biosynthesis. As used herein, the term "biosynthesis" means any method utilizing a translation system (cellular or noncellular), including the use of at least one of the following components: polynucleotides, codons, tRNA, and ribosomes. As an example, non-natural amino acids can be "biosynthetically incorporated" into non-natural amino acid peptides using methods and techniques described herein and well known in the art. See, for example, WO2010 / 011735 and WO2005 / 074650.

[0081] The term "conservatively modified variant" applies to natural and non-natural amino acids, as well as natural and non-natural nucleic acid sequences and combinations thereof. With respect to a particular nucleic acid sequence, a "conservatively modified variant" refers to those natural and non-natural nucleic acids that encode the same or substantially the same natural and non-natural amino acid sequence, or substantially the same sequence where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence. As an example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where the codon specifies alanine, the codon can be changed to any of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variants," which are variations of conserved modification. Therefore, by way of example, each natural or non-natural nucleic acid sequence encoding a natural or non-natural polypeptide herein also describes each possible silent variant of the natural or non-natural nucleic acid. Those skilled in the art will recognize that each codon in a natural or non-natural nucleic acid (except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent variation of the natural and non-natural nucleic acids encoding natural and non-natural polypeptides is implicit in each of the described sequences. Regarding the amino acid sequence, alterations, additions, or deletions of a single natural or non-natural amino acid or a small percentage of natural and non-natural amino acids in the encoded sequence of a nucleic acid, peptide, polypeptide, or protein are “variants of conserved modification” where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural or non-natural amino acid with a chemically similar amino acid. Providing a conserved representation of a functionally similar natural amino acid is well known in the art. Providing a conserved representation of a functionally similar amino acid is known to those skilled in the art. The following eight groups each contain amino acids that are conserved in their substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd ed., 1993). Variations of such conserved modifications are complementary to, and not excluded from, the polymorphic variants, interspecific homologs, and alleles of the compositions described herein.

[0082] As used herein, the term "medicine" refers to any substance intended to prevent, diagnose, alleviate, treat, or cure a disease or ailment, such as cancer, including prostate cancer.

[0083] As used herein, the term "effective amount" refers to an adequate quantity of an applied agent, compound, or composition that will alleviate, to a certain extent, one or more symptoms of the disease or condition being treated. The result may be a reduction and / or mitigation of signs, symptoms, or the cause of the disease, or any other desired change in the biological system. As examples, the applied agent, compound, or composition includes, but is not limited to, natural amino acid peptides, non-natural amino acid peptides, modified natural amino acid peptides, modified non-amino acid peptides, or antibodies or variants thereof. Compositions containing such natural amino acid peptides, non-natural amino acid peptides, modified natural amino acid peptides, modified non-natural amino acid peptides, or antibodies or variants thereof may be applied for prophylactic, enhancing, and / or therapeutic treatment. The appropriate "effective" amount in any case may be determined using techniques such as dose escalation studies.

[0084] The term “enhance” means to increase or prolong the potency or duration of a desired effect. For example, “enhancing” the effect of a therapeutic agent refers to the ability to increase or prolong the potency or duration of the agent’s effect during treatment of a disease, condition, or symptom. As used herein, “enhancing effective amount” refers to an amount sufficient to enhance the therapeutic effect of the agent in treating a disease, condition, or symptom. When used in a patient, the effective amount for that purpose will depend on the severity and course of the disease, condition, or symptom, prior treatment, the patient’s health status and response to the drug, and the judgment of the treating physician.

[0085] As used herein, the term "half-life" refers to the time required for any specified property to decrease by half. Typically, a specified property is the concentration of a substance in the body or a body compartment, wherein the substance is a conjugated protein of this disclosure, or a corresponding protein or its unconjugated equivalent. The term "half-life" may also be referred to herein as "t". 1 / 2 "or "T 1 / 2 ".

[0086] As used herein, the term "elimination half-life" refers to a pharmacokinetic parameter defined as the time period in which the concentration of a biologic in a subject's plasma or serum, or in the total amount in the subject's whole body, decreases by approximately 50%. Therefore, after one half-life, the concentration of the biologic in the subject's plasma or serum, or in the whole body, will be half of the initial concentration. Typically, the time period in which the concentration of the biologic (e.g., in the blood, plasma, or serum, or in the whole body) decreases by approximately 50% begins at or around the time the biologic is administered to the subject.

[0087] As used herein, the term “terminal half-life” refers to the time required to divide the serum or plasma concentration by two after reaching pseudo-equilibrium, rather than the time required to eliminate half of the administered dose. See, for example, Toutain PL and Bousquet-Melou A. (2004) J. Vetorical Pharmacology and Therapeutics, 27(6):427-439.

[0088] The term "humanized or chimeric antibody" refers to a molecule typically prepared using recombinant techniques that has an antigen-binding site derived from an immunoglobulin of a non-human species (e.g., mouse), and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. Generally, a humanized antibody will contain at least one, and typically two, variable domains, where all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin, and all or substantially all of the framework residues / regions (FRs) are framework residues / regions of the human immunoglobulin sequence. A humanized antibody will also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin. Humanized forms of rodent antibodies will substantially contain the same CDR sequence as the parent rodent antibody, although certain amino acid substitutions may be included to increase affinity, increase the stability of the humanized antibody, or for other reasons. However, since CDR loop exchange does not consistently produce antibodies with the same binding properties as the source antibody, variations in framework residues (FRs) (residues involved in CDR loop support) can also be introduced into humanized antibodies to maintain antigen-binding affinity. The antigen-binding site can contain an intact variable domain fused to a constant domain or simply a complementarity-determining region (CDR) transplanted onto an appropriate framework region within the variable domain. The antigen-binding site can be wild-type or modified by substitution of one or more amino acids. This eliminates the constant region as an immunogen in the human individual, but the possibility of an immune response to the exogenous variable region remains (LoBuglio, AF et al., “Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224, 1989). Another approach focuses not only on providing a human-derived constant region but also on modifying the variable region to reshape it as closely as possible to the human morphology. Both the heavy and light chains have known variable regions containing three complementarity-determining regions (CDRs) that vary in response to the antigen in question and determine binding capacity, flanked by four framework regions (FRs), which are relatively conserved in a given species and are assumed to provide scaffolding for the CDRs. When preparing nonhuman antibodies against a specific antigen, the variable regions can be "humanized" by grafting CDRs derived from the nonhuman antibody onto the FRs present in the human antibody to be modified.The application of this method to various antibodies has been reported in the following literature: Kettleborough, CA et al., “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation,” Protein Engineering 4:773-3783, 1991; Co, MS et al., “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Acad. Sci. (USA) 88:2869-2873, 1991; Carter, P. et al., “Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289, 1992; and Co, MS et al., “Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen,” J. Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, which are also referred to as "derived from" one or more CDRs from one or more CDRs from the original antibody.

[0089] As used herein, the term "identical" refers to two or more identical sequences or subsequences. Furthermore, the term "substantially identical" as used herein means that two or more sequences have a certain percentage of identical contiguous units when compared and aligned within a comparison window or specified region to obtain maximum correspondence, as measured by comparison algorithms or by manual alignment and visual inspection. By way of example only, two or more sequences may be considered "substantially identical" if contiguous units are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical within a specified region. Such percentages describe the "percentage of identity" of two or more sequences. Sequence identity can exist within regions of at least approximately 75 to 100 contiguous units, within regions of approximately 50 contiguous units, or throughout the sequence if not specified. This definition also refers to complementary sequences of the test sequences. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, and are "substantially identical" if the amino acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical within a specified region. Identity may exist in a region of at least about 75 to about 100 amino acids, in a region of about 50 amino acids, or throughout the entire sequence of the polypeptide if not specified. Furthermore, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, and are "substantially identical" if the nucleic acid residues are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical within a specified region. Identity may exist in regions of at least about 75 to about 100 nucleic acids in length, in regions of about 50 nucleic acids in length, or throughout the entire sequence of a polynucleotide sequence if not specified.

[0090] As used herein, the term "immunogenicity" refers to an antibody response to the administration of a therapeutic agent. Immunogenicity of therapeutic non-natural amino acid peptides can be obtained using quantitative and qualitative assays for detecting antibodies against non-natural amino acid peptides in biological fluids. Such assays include, but are not limited to, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), luminescent immunoassay (LIA), and fluorescence immunoassay (FIA). Analysis of the immunogenicity of therapeutic non-natural amino acid peptides involves comparing the antibody response upon administration of the therapeutic non-natural amino acid peptide with the antibody response upon administration of the therapeutic natural amino acid peptide.

[0091] As used herein, the term "isolated" means the component of interest has been separated from and removed from a component of no interest. The isolated substance may be in a dry or semi-dry state, or in solution, including but not limited to aqueous solutions. The isolated component may be in a homogeneous state, or the isolated component may be part of a pharmaceutical composition containing additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity may be determined using analytical chemistry techniques, including but not limited to polyacrylamide gel electrophoresis or high-performance liquid chromatography. Furthermore, when the component of interest is isolated and is the main substance present in the formulation, the component is described herein as substantially purified. The term "purified" as used herein may refer to a component of interest with a purity of at least 85%, at least 90%, at least 95%, at least 99%, or higher. By way of example only, a nucleic acid or protein is "isolated" when it does not contain at least some cellular components to which it is bound in its native state, or when the nucleic acid or protein has been concentrated to a level higher than its in vivo or in vitro production concentration. Furthermore, for example, a gene is isolated when it is isolated from an open reading frame flanking a gene that encodes a protein different from the gene of interest.

[0092] As used herein, the term "bond" refers to a bond or chemical moiety formed by a chemical reaction between a functional group of a linker and another molecule. Such bonds can include, but are not limited to, covalent and non-covalent bonds, while such chemical moiety can include, but is not limited to, esters, carbonates, imines, phosphates, hydrazones, acetals, orthoesters, peptide bonds, and oligonucleotide bonds. Hydrolytically stable bonds are those that are substantially stable in water and do not react with water at a useful pH, including but not limited to, not reacting with water for extended periods under physiological conditions, and possibly even indefinitely. Hydrolytically unstable or degradable bonds mean that the bond is degradable in water or aqueous solutions, including, for example, blood. Enzymatically unstable or degradable bonds mean that the bond can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable bonds in the polymer backbone or in the linking groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. Such degradable bonds include, but are not limited to, ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups typically hydrolyze under physiological conditions to release the bioactive agent. Other hydrolyzable bonds include, but are not limited to, carbonate bonds; imine bonds formed by the reaction of amines and aldehydes; phosphate bonds formed by the reaction of alcohols with phosphate groups; hydrazone bonds as products of the reaction of hydrazides and aldehydes; acetal bonds as products of the reaction of aldehydes and alcohols; orthoester bonds as products of the reaction of formates and alcohols; peptide bonds formed by, but not limited to, amine groups at the ends of polymers such as PEGs and carboxyl groups of peptides; and oligonucleotide bonds formed by, but not limited to, phosphoramidoid groups at the ends of polymers and 5' hydroxyl groups of oligonucleotides.

[0093] As used herein, the term "metabolite" refers to a derivative of a compound (e.g., a natural amino acid peptide, a non-natural amino acid peptide, a modified natural amino acid peptide, or a modified non-natural amino acid peptide) formed during the metabolism of that compound. The terms "pharmaceutical active metabolite" or "active metabolite" refer to a bioactive derivative of a compound (e.g., a natural amino acid peptide, a non-natural amino acid peptide, a modified natural amino acid peptide, or a modified non-natural amino acid peptide) formed during the metabolism of that compound.

[0094] As used herein, the term "metabolic" refers to an overview of the processes by which a particular substance is altered by an organism. Such methods include, but are not limited to, hydrolysis and enzyme-catalyzed reactions. Further information on metabolism is available in The Pharmacological Basis of Therapeutics, 9th edition, McGraw-Hill (1996). By way of example only, metabolites of natural amino acid peptides, non-natural amino acid peptides, modified natural amino acid peptides, or modified non-natural amino acid peptides can be identified by applying the natural amino acid peptide, non-natural amino acid peptide, modified natural amino acid peptide, or modified non-natural amino acid peptide to a host and analyzing tissue samples from the host, or by incubating the natural amino acid peptide, non-natural amino acid peptide, modified natural amino acid peptide, or modified non-natural amino acid peptide in vitro with hepatocytes and analyzing the resulting compounds.

[0095] As used herein, the term "modified" refers to any alteration to a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such alterations or modifications may be obtained through post-synthetic modification of the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, or through co-translational or post-translational modification of the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide.

[0096] "Non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrolysine, or selenocysteine. Other terms that may be used synonymously with "non-natural amino acid" are "non-naturally encoded amino acid," "non-natural amino acid," "non-naturally occurring amino acid," and their various hyphenated and unhyphenated forms. The term "non-natural amino acid" includes, but is not limited to, amino acids that are naturally occurring but not incorporated into the grown polypeptide chain by modifying naturally encoded amino acids (including, but not limited to, the 20 common amino acids, or pyrrolidone and selenocysteine). Examples of naturally occurring non-naturally encoded amino acids include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine. Additionally, the term "non-natural amino acid" includes, but is not limited to, amino acids that are not naturally occurring and can be obtained synthetically or by modifying non-natural amino acids.

[0097] As used herein, the term “nucleic acid” refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides in single-stranded or double-stranded form, as well as polymers thereof. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides; (ii) oligonucleotide analogs, including but not limited to PNAs (peptide nucleic acids), DNA analogs (phosphate thioides, aminophosphates, etc.) used in antisense techniques; and (iii) conserved variants of them (including but not limited to degenerate codon substitutions) as well as complementary sequences and explicitly indicated sequences. As an example, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixture of bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0098] As used herein, the term “pharmaceutically acceptable” means a material that does not eliminate the biological activity or properties of a compound and is relatively non-toxic, including but not limited to salts, binders, adjuvants, excipients, carriers, or diluents; that is, the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any component of a composition containing the material.

[0099] In some embodiments, the present invention relates to polymers. As used herein, the term "polymer" refers to a molecule composed of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides or polysaccharides, or polyalkylene glycols. The polymers of the present invention can be linear or branched polymeric polyether polyols, including but not limited to polyethylene glycol, polypropylene glycol, polybutanediol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogues such as Shearwater Corporation's catalogue "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymers have an average molecular weight of about 0.1 kDa to about 100 kDa. Such polymers include, but are not limited to, about 100 Da to about 100,000 Da or more. The molecular weight of the polymer can be from about 100 Da to about 100,000 Da, including but not limited to about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, and about 30,000 Da. The molecular weights of the polymers are approximately 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is from about 100 Da to about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 1,000 Da to about 40,000 Da. In some embodiments, the polymer has a molecular weight of about 2,000 Da to about 50,000 Da. In some embodiments, the polymer has a molecular weight of about 5,000 Da to about 40,000 Da. In some embodiments, the polymer has a molecular weight of about 10,000 Da to about 40,000 Da. In some embodiments, the poly(ethylene glycol) molecule is a branched polymer.The molecular weight of branched PEG can be from about 1,000 Da to about 100,000 Da, including but not limited to about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, and about 45,000 Da. The molecular weights of branched PEG are approximately 0 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of branched PEG is from about 1,000 Da to about 50,000 Da. In some embodiments, the molecular weight of branched PEG is from about 1,000 Da to about 40,000 Da. In some embodiments, the molecular weight of branched PEG is from about 5,000 Da to about 40,000 Da. In some embodiments, the molecular weight of branched PEG is from about 5,000 Da to about 20,000 Da. In other embodiments, the branched PEG has a molecular weight of about 2,000 Da to about 50,000 Da. The terms "PEGylating" or "PEGylated" refer to the covalent bonding of a specific synthetic amino acid to a polyethylene glycol (PEG) molecule. The method may include contacting a separated α-PSMA ADC peptide containing the synthetic amino acid with a water-soluble polymer containing a portion that reacts with the synthetic amino acid.

[0100] The terms “polypeptide,” “peptide,” or “protein” are used interchangeably herein and refer to a polymer of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. This term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids. Furthermore, such “polypeptides,” “peptides,” and “proteins” include amino acid chains of any length, including full-length proteins, where amino acid residues are linked by covalent peptide bonds.

[0101] The term "post-translational modification" refers to any modification of a natural or non-natural amino acid that occurs after that amino acid has been translated and incorporated into the polypeptide chain. Such modifications include, but are not limited to, in vivo co-translational modifications, in vitro co-translational modifications (such as in cell-free translation systems), in vivo post-translational modifications, and in vitro post-translational modifications.

[0102] As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to a pharmaceutical preparation that is converted into the parent drug in vivo or in vitro without eliminating the biological activity or properties of the drug and is relatively non-toxic. That is, the material can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any component of a composition containing the material. Prodrugs are typically drug precursors that, upon administration to a subject and subsequent absorption, are converted into the active or more active substance via processes such as conversion through metabolic pathways. Some prodrugs have chemical groups present on the prodrug that reduce its activity and / or impart drug solubility or other properties. Once the chemical groups have been cleaved from and / or modified on the prodrug, the active drug is produced. Prodrugs are converted into the active drug in vivo through enzymatic or non-enzymatic reactions. Prodrugs can provide improved physiological and chemical properties, such as better solubility, enhanced delivery properties, such as specific targeting of specific cells, tissues, organs, or ligands, and improved therapeutic value of the drug. The beneficial effects of such prodrugs include, but are not limited to, (i) ease of administration compared to the parent drug; (ii) bioavailability via oral administration, which the parent drug cannot; and (iii) improved solubility in a pharmaceutical composition compared to the parent drug. Prodrugs include pharmacologically inactive or less active derivatives of active pharmaceutical ingredients. Prodrugs can be designed to modulate the amount of drug or bioactive molecule reaching a desired site of action by manipulating properties of the drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. A non-limiting example of a prodrug is a non-natural amino acid polypeptide administered as an ester (“prodrug”) to facilitate transport across water-soluble, mobility-detrimental cell membranes, which, once inside the water-soluble, beneficial cells, are metabolically hydrolyzed to a carboxylic acid (the active entity). Prodrugs can be designed as reversible drug derivatives to act as modifiers that enhance drug transport to site-specific tissues.

[0103] As used herein, the term "preventative effective dose" refers to the amount of a composition containing at least one non-natural amino acid polypeptide or at least one modified non-natural amino acid polypeptide that is administered prophylactically to a patient, which will alleviate to some extent one or more symptoms of the disease, condition, or symptom being treated. In such preventative applications, this dose may depend on the patient's health condition, weight, etc. Determining such preventative effective doses through routine experiments (including, but not limited to, dose-escalation clinical trials) is considered well known to those skilled in the art.

[0104] The term "recombinant host cell," also known as "host cell," refers to a cell containing exogenous polynucleotides, wherein the methods for inserting the exogenous polynucleotides into the cell include, but are not limited to, direct uptake, transduction, f-crossing, or other methods known in the art for generating recombinant host cells. By way of example only, such exogenous polynucleotides may be non-integrating vectors, including but not limited to plasmids, or may be integrated into the host genome.

[0105] As used herein, the term "subject" refers to an animal used as a subject of treatment, observation, or experimentation. By way of example only, a subject can be, but is not limited to, mammals (including, but not limited to, humans). The terms "subject" and "patient" are used interchangeably herein.

[0106] As used herein, the term "substantially purified" means a component of interest that is substantially free of other components that typically accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" when a formulation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (on a dry weight basis) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher. By way of example only, natural or non-natural amino acid peptides may be purified from native cells, or, in the case of recombinant natural or non-natural amino acid peptides, from host cells. As an example, when the formulation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminants, the formulation of natural or non-natural amino acid peptides may be "substantially purified". As an example, when the natural or non-natural amino acid peptides are recombinantly generated from host cells, the natural or non-natural amino acid peptides may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the cell dry weight. As an example, when natural or non-natural amino acid peptides are recombinantly generated from host cells, the natural or non-natural amino acid peptides may be present in the culture medium at concentrations of about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of the cell dry weight. As an example, “substantially purified” natural or non-natural amino acid peptides may have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or higher, as determined by appropriate methods, including but not limited to SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0107] As used herein, the term "therapeutic effective amount" refers to an amount of a composition containing at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide administered to a patient who already has a disease, symptom, or condition, sufficient to cure or at least partially inhibit or alleviate one or more symptoms of the treated disease, symptom, or condition. The effectiveness of such compositions depends on conditions, including but not limited to the severity and duration of the disease, symptom, or condition, prior treatment, the patient's health status and response to the drug, and the judgment of the treating physician. By way of example only, the therapeutic effective amount can be determined through routine experiments, including but not limited to dose-escalation clinical trials.

[0108] As used herein, the terms “toxicity,” “toxic moiety,” “toxic group,” “cytotoxic,” “cytotoxic payload,” “payload,” or “free payload” refer to compounds that can cause injury, disorder, or death. Terms including toxic moiety (including payload and free payload) include, but are not limited to: auristatin, amberstatin 269 (AS269), DNA minor groove binder, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, taxane, puromycin, dolastatin, maytansinoids, and vinca alkaloids. Alkaloid), AFP, Monomethyl olprestatin F (MMAF), MMAE, AEB, AEB, olprestatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, sea hare toxin 10, echinomycin, compretatstatin, chalicheamicin, maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide, teniposide) Poside, etc.), baccatin and its derivatives, anti-microtubule agents, cryptophysin, comprbetastatin, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, epothilone A, epothilone B, nocodazole, colchicine, colcimid, estramustine, cemadotin, discormolide, maytansine, eleutherobin, mechlorethamine.Cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipebroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, temozolomide, ytarabine, cytosine arabinoside arabinoside, fluorouracil, fluxuridine, 6-thioguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydrofolate, leucovorin, fludarabine phosphate Phosphate), pentostatin, gemcitabine, Ara-C, deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine, brequinar, and antibiotics (e.g., anthracycline, gentamicin, cefalotin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, rocithromycin, furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacillin, methicillin, penicillin).Ciprofloxacin, moxifloxacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaximin, etc., and antiviral drugs (e.g., abacavir, acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavire) Entecavir, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valacyclovir, zanamivir, etc., daunorubicin hydrochloride. Hydrochloride, daunomycin, rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin, and morpholino derivatives, phenoxazinone bicyclic peptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., plicamycin, mithramycin), anthraquinones (e.g., mitoxantrone), aziridine pyrroloindole-diones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf).Rapamycin, navelbene, CPT-11, anastrozole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, droloxafine, allocolchicine, halichondrin B, colchicine, colchicine derivatives, maytansine, rhizomycin, palitase, palitase derivatives, docetaxel, thiocolchicine, trimethylcysteine, vinblastine sulfate, vincristine sulfate, cisplatin, carboplatin, hydroxyurea, N-methylhydrazine (Nm ethylhydrazine), epidophyllotoxin, procarbazine, mitoxantrone, formyltetrahydrofolate, and tegafur; and any of the foregoing is further conjugated to a linker and / or an amino acid, including but not limited to non-natural amino acids. For example, the term "payload" or "free payload" may refer to a compound that can cause injury, interference, or death, wherein the compound comprises or is composed of a toxic moiety further conjugated to a linker and / or a non-natural amino acid. As described herein, a free payload can be released from the anti-PSMA ADC of this disclosure in vivo after administration to a subject. For example, as disclosed herein, the free payload pAF-AS269 is released from the anti-PSMA ADC ARX517. As disclosed in Example 12, once ARX517 is internalized by cancer cells, ARX517 undergoes proteolytic degradation and releases the free payload pAF-AS269, which has, Figure 8C The structure described herein comprises a cytotoxic moiety AS269 conjugated to the non-natural amino acid p-acetylphenylalanine, wherein the non-natural amino acid is derived from the heavy chain amino acid sequence of the ARX517 antibody. Therefore, in some embodiments, the "free payload" of this disclosure is pAF-AS269.

[0109] The term "taxane" includes palitaxetine, as well as any active taxane derivatives or prodrugs.

[0110] As used herein, the term "treatment" includes relieving, preventing, reducing, or improving symptoms of a disease or condition; preventing additional symptoms; improving or preventing the underlying metabolic cause of symptoms; inhibiting a disease or condition, such as preventing its progression; alleviating a disease or condition; causing a disease or condition to subside; relieving the condition caused by a disease or condition; or stopping the symptoms of a disease or condition. The term "treatment" includes, but is not limited to, preventative and / or therapeutic treatment. The term "treatment" may also refer to reducing, alleviating, or improving one or more symptoms associated with prostate cancer.

[0111] As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in aqueous solvents. Such water-soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, and their mono-C1-C2 polymers. 10 Alkoxy or aryloxy derivatives (described in U.S. Patent 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, polysaccharides, cellulose and cellulose derivatives (including, but not limited to, methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, polyvinyl ethyl ether and α-β-poly[(2-hydroxyethyl)-DL-asparagine, etc., or mixtures thereof. By way of example only, conjugation of such water-soluble polymers with natural or non-natural amino acid peptides can lead to changes, including but not limited to increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, modulated biological activity, prolonged circulation time, modulated immunogenicity, modulated physical characteristics (including but not limited to aggregation and multimerization), altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. Furthermore, such water-soluble polymers may or may not possess their own biological activity.

[0112] As used herein, the term "regulated serum half-life" refers to a positive or negative change in the cyclic half-life of a modified bioactive molecule relative to its unmodified form. As an example, modified bioactive molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid peptides, or non-natural amino acid peptides. As an example, serum half-life is measured by taking blood samples at different time points after administration of the bioactive molecule or modified bioactive molecule and determining the concentration of the molecule in each sample. The correlation between serum concentration and time allows for the calculation of serum half-life. As an example, regulated serum half-life can be a prolongation of serum half-life, which can enable improved dosing regimens or avoid toxic effects. Such an increase in serum half-life can be at least about two-fold, at least about three-fold, at least about five-fold, or at least about ten-fold. Methods for assessing serum half-life are known in the art and can be used to assess the serum half-life of the antibodies and antibody-drug conjugates of the present invention.

[0113] As used herein, “taxane” is an anticancer agent that interferes with or disrupts microtubule stability, formation, and / or function. Such agents include palitaxetine, docetaxel, and cabazitaxel, as well as any prodrug or active derivative of any of the foregoing, wherein these derivatives act on microtubules through the same mode of action as the taxanes from which they are derived. In some embodiments, “taxane” refers to one or more of the following: palitaxetine, docetaxel, and cabazitaxel.

[0114] As used herein, the term "regulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified bioactive molecule relative to its unmodified form. As examples, modified bioactive molecules include, but are not limited to, natural amino acids, non-natural amino acids, natural amino acid peptides, or non-natural amino acid peptides. As an example, therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at different time points after administration. An extended therapeutic half-life can enable specific beneficial dosing regimens, specific beneficial total doses, or avoid undesirable effects. As an example, an extended therapeutic half-life can result from increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased other parameters or mechanisms of action of the unmodified molecule, or increased or decreased degradation of the molecule by an enzyme (such as, by way of example only, a protease). Methods for assessing therapeutic half-life are known in the art and can be used to assess the therapeutic half-life of the antibodies and antibody-drug conjugates of the present invention.

[0115] Antibody-based therapies have become an important component of the treatment of an increasing number of human malignancies in fields such as oncology, inflammatory diseases, and infectious diseases. In most cases, the therapeutic function is based on the high specificity and affinity of antibody-based drugs for their target antigens. Equipping monoclonal antibodies with drugs, toxins, or radionuclides is another strategy for monoclonal antibodies to induce therapeutic effects. By combining the precise targeting specificity of antibodies with the tumor-killing capabilities of toxic effector molecules, immunoconjugates allow for sensitive differentiation between target and normal tissues, resulting in fewer side effects than most conventional chemotherapy drugs. The toxins used can specifically, stably, and irreversibly conjugate to unique sites on the antibody. This unique conjugation process allows for precise control over the location of the toxin on the antibody and the number of toxins conjugated to each antibody. These two characteristics are crucial for controlling the biophysical properties and toxicity associated with ADCs (see, for example, Jackson et al., 2014; Tian et al., 2014).

[0116] The anti-PSMA antibody pharmaceutical conjugates provided in this disclosure include humanized or chimeric monoclonal antibodies and variants that bind to the extracellular domain of prostate-specific membrane antigen (PSMA). Prostate-specific membrane antigen is a type II membrane protein highly expressed, for example, in prostate intraepithelial neoplasia (PIN), primary prostate cancer, and metastatic prostate cancer. The anti-PSMA antibodies disclosed herein can be any known PSMA antibody having at least one non-naturally encoded or unnaturally encoded amino acid.

[0117] In one embodiment, the present invention provides anti-PSMA antibodies having non-naturally encoded amino acids, antibody fragments, and variants thereof, which facilitate conjugation of the antibody to a drug (e.g., a drug, a toxin molecule). In one embodiment, the ADC comprises an anti-PSMA antibody conjugated to a drug, wherein the conjugation occurs via non-naturally encoded amino acids in the antibody. In one embodiment, the ADC comprises an anti-PSMA antibody conjugated to a drug, wherein the conjugation occurs via non-naturally encoded amino acids in the heavy chain of the antibody. In one embodiment, the ADC comprises an anti-PSMA antibody conjugated to a drug, wherein the conjugation occurs via non-naturally encoded amino acids in the light chain of the antibody. In one embodiment, the ADC comprises a full-length antibody conjugated to a drug, wherein the conjugation occurs via non-naturally encoded amino acids in the antibody. In one embodiment, the ADC comprises a full-length antibody conjugated to a drug, wherein the conjugation occurs via non-naturally encoded amino acids in the light chain of the antibody.

[0118] In some embodiments, the ADC is a cytotoxic drug or agent. In some aspects of the invention, the cytotoxic drug is selected from the group consisting of: aurestatin, DNA minor groove binding agents, DNA minor groove alkylating agents, enediyne, lecithin, pyroxine, taxane, puromycin, sea hare toxin, maytansine derivatives, and vinca alkaloids. In some aspects of the invention, the cytotoxic drug is AFP, MMAF, MMAE, AEB, AEB, aurestatin E, palitaxetine, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizomycin, cyanomorpholino-doxorubicin, sea hare toxin 10, echinomycin, cobustatin, chalcoxam, maytansine, DM-1, or fusiformin, but is not limited thereto.

[0119] In some aspects of the invention, the cytotoxic agent is an anti-tubulin agent. In some embodiments, the anti-tubulin agent is aurestatin, vinca alkaloids, podophyllotoxin, taxane, berry gibberellin derivatives, cryptophytic acid, maytansine, cobustatin, or sea haretoxin. In other aspects of the invention, the anti-tubulin agent is AFP, MMAF, MMAE, AEB, AEB, aurestatin E, vincristine, vinblastine, vinorelbine, VP-16, camptothecin, palitaxetine, docetaxel, epokycin A, epokycin B, nocodazole, colchicine, colchicineamine, estradiol, simadalide, scutellarin, maytansine, DM-1, or soft coral alcohol, but is not limited thereto.

[0120] In other aspects of the invention, the cytotoxic agent of the ADC is ganciclovir, etanercept, cyclosporine, tacrolimus, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, cortisol, aldosterone, dexamethasone, cyclooxygenase inhibitor, 5-lipoxygenase inhibitor, or leukotriene receptor antagonist.

[0121] In some embodiments of the present invention, the antibody of the ADC comprises a full-length antibody or a fragment thereof that binds to PSMA and is conjugated to a cytotoxic agent or immunosuppressant, wherein the antibody-drug conjugate exerts: (a) cytotoxic or cytosuppressive effects on cancer cell lines expressing PSMA, or (b) cytotoxic, cytosuppressive, or immunosuppressive effects on immune cells expressing PSMA, wherein the conjugation occurs at a non-naturally encoded amino acid in the antibody.

[0122] In some embodiments, the antibodies, variants, or compositions disclosed herein may be antibodies, variants, or compositions that bind to the PSMA receptor. In other embodiments of the invention, the antibodies, variants, or compositions may be antibodies, variants, or compositions that bind to the extracellular surface of the PSMA receptor. In yet another embodiment of the invention, the disclosed antibodies, variants, or compositions may be antibodies, variants, or compositions that bind to PSMA dimers. In some embodiments, the antibodies, variants, or compositions disclosed herein may be antibodies, variants, or compositions having a CDR from J591 grafted onto a frame region of the variable region. In other embodiments, the antibodies, variants, or compositions disclosed herein may be antibodies, variants, or compositions having non-naturally encoded amino acids. In some embodiments, the antibodies, variants, or compositions may be antibodies, variants, or compositions described elsewhere in this disclosure. In some embodiments, the antibodies, antibody variants, or antibody compositions disclosed herein may be fully humanized. In other embodiments, the antibodies, antibody variants, or antibody compositions disclosed herein may be chimeric. In some embodiments of the present invention, the antibody may be a full-length antibody (variable region + Fc region), Fab, bispecific, Fab dimer, Fab bispecific, Fab trispecific, bispecific T cell conjugate, biaffinity retargeting antibody, IgG1 / IgG3 bispecific antibody, bivalent antibody, bispecific biantibody, scFv-Fc, or microantibody.

[0123] Methods, compositions, and techniques for producing and using sea haretoxin linker derivatives or analogs comprising at least one carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketamine, ketalynyl, alkyne, cycloalkyne, or enedione are well known to those skilled in the art (see, for example, WO2013 / 185117, the entire text of which is incorporated herein by reference). Methods, compositions, and techniques for producing and using sea haretoxin linker derivatives or analogs comprising at least one non-natural amino acid or modified non-natural amino acid having an oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.) are also well known to those skilled in the art and are described, for example, in WO2013 / 185117, the entire text of which is incorporated herein by reference. Such sea haretoxin linker derivatives containing non-natural amino acids may contain other functional groups, including but not limited to: polymers; water-soluble polymers; polyethylene glycol derivatives; second proteins or peptides or peptide analogs; antibodies or antibody fragments; and any combination thereof. It should be noted that the various aforementioned functional groups do not imply that a member of one functional group cannot be classified as a member of another. In fact, overlap will exist depending on the specific circumstances. For example, water-soluble polymers overlap in scope with polyethylene glycol derivatives; however, the overlap is not complete, and therefore both functional groups are mentioned above.

[0124] In some embodiments, this document provides toxic group adapter derivatives comprising carbonyl, dicarbonyl, oxime, hydroxylamine, amino-oxygen, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketone-amine, ketone-alkyne, alkyne, cycloalkyne, or ene-dione. In some embodiments, the toxic group derivative comprises any of the adapters disclosed herein. Methods, compositions, and techniques for generating and using toxic group derivatives or analogs comprising at least one non-natural amino acid or modified non-natural amino acid having an oxime, aromatic amine, heterocycle (e.g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.) are described in WO2013 / 185117 (the entire text of which is incorporated herein by reference). In some embodiments, such toxic derivatives containing non-natural amino acids may contain additional functional groups, including but not limited to: polymers; water-soluble polymers; polyethylene glycol derivatives; second proteins or peptides or peptide analogs; antibodies or antibody fragments; and any combination thereof. In a specific embodiment, the toxic group is a tubulin inhibitor. In some specific embodiments, the toxic group is salipodin or aurestatin. In other specific embodiments, the toxic group is salipodin or an aurestatin derivative. It should be noted that the various aforementioned functional groups do not imply that a member of one functional group cannot be classified as a member of another functional group. In fact, overlap will exist depending on the specific circumstances. By way of example only, water-soluble polymers overlap in scope with polyethylene glycol derivatives; however, the overlap is not complete, and therefore both functional groups are referred to above.

[0125] Some embodiments of the present invention describe the preparation of certain toxic moieties having a linker that reduces the toxicity of the moieties in vivo while retaining their pharmacological activity. In some embodiments, when administered to animals or humans, the toxicity of the linked toxic group is reduced or eliminated compared to a free toxic group or a derivative of a toxic group containing an unstable bond, while retaining pharmacological activity. In some embodiments, increased doses of the linked toxic group (e.g., sarsatoxin linker derivatives, non-natural amino acid-linked sarsatoxin derivatives) can be administered to animals or humans with greater safety. In some embodiments, non-natural amino acid peptides linked to the pharmaceutical moieties (e.g., sarsatoxin derivatives) provide in vitro and in vivo stability. In some embodiments, non-natural amino acid peptides linked to toxic moieties (e.g., sarsatoxin-10 derivatives) are more effective and less toxic than free toxic moieties (e.g., tubulin inhibitors, sarsatoxin-10).

[0126] This invention provides anti-PSMA antibody-pharmaceutical conjugates (ADCs) and pharmaceutical formulations containing them. More specifically, this disclosure relates to methods and uses of anti-PSMA ADCs and pharmaceutical compositions containing them in the inhibition, prevention, or treatment of PSMA-related diseases or cancers. In some embodiments, the cancer is prostate cancer or metastatic castration-resistant prostate cancer. In some embodiments, the method includes administering an anti-PSMA ADC of this disclosure, such as ARX517, to a subject. Pharmaceutical formulations containing an effective amount of the anti-PSMA ADC and being storage-stable are also provided.

[0127] Methodology and Technology This disclosure covers methods and techniques well known in the art. These methods include conventional methods in mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, which are within the scope of the art. The compounds disclosed herein can be synthesized using several methods or schemes employed in the art. See, for example, Dubowchik et al., Bioconjugate Chem. 13: 855-869, 2002; Doronina et al., Nature Biotechnology 21(7):778-784, 2003; WO2012 / 166560; WO2013 / 185117, each of which is incorporated herein by reference. Many methodologies and techniques for synthesizing pharmaceutical, diagnostic, or therapeutic compounds are well known to those skilled in the art.

[0128] Unless otherwise stated, this invention also covers conventional techniques in molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, all of which are within the scope of the art. These techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY* (edited by Sambrook et al., 2001); *Oligonucleotide Synthesis: Methods And Applications (Methods in Molecular Biology), edited by Herdewijn, P., Humana Press, Totowa, NJ; *Oligonucleotide Synthesis* (edited by Gait, MJ, 1984); *Methods In Molecular Biology*, Humana Press, Totowa, NJ; *Cell Biology: A Laboratory Notebook*, Academic Press, New York, NY (edited by Cellis, JE, 1998); *Animal Cell Culture* (edited by Freshney, RI, 1987); *Introduction To Cell And Tissue Culture*, Plenum Press, New York, NY (edited by Mather, JP and Roberts, PE, 1998); *Cell And Tissue Culture: Laboratory Procedures* John Wiley and Sons, Hoboken, NJ, (eds. Doyle, A. et al., 1993-8); Methods In Enzymology (Academic Press, Inc.) New York, NY; Weir's Handbook Of Experimental Immunology Wiley-Blackwell Publishers, New York, NY, (Herzenberg, LA(Edited by Miller, JM, et al., 1997); Gene Transfer Vectors For Mammalian Cells Cold Spring Harbor Press, Cold Spring Harbor, NY, (Edited by Miller, JM, et al., 1987); Current Protocols In Molecular Biology, Greene Pub. Associates, New York, NY, (Edited by Ausubel, FM, et al., 1987); PCR: The Polymerase Chain Reaction, Birkhauser, Boston, MA, (Edited by Mullis, K., et al., 1994); Current Protocols In Immunology, John Wiley and Sons, Hoboken, NJ, (Edited by Coligan, JE, et al., 1991); Short Protocols In Molecular Biology, Hoboken, NJ, (John Wiley and Sons, 1999); Immunobiology 7 Garland Science, London, UK, (Edited by Janeway, CA, et al., 2007); Antibodies. Stride Publications, Devoran, UK, (P. Finch, 1997); Antibodies: A Practical Approach Oxford University Press, USA, New York, NY, (edited by D. Catty., 1989); Monoclonal Antibodies: A Practical Approach Oxford University Press, USA, New York NY, (Editors such as Shepherd, P. et al., 2000); Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press Harbor,NY, (Harlow, E.Edited by Zanetti et al., 1998; The Antibodies Harwood Academic Publishers, London, UK, (edited by Zanetti, M. et al., 1995).

[0129] Sea haretoxin connector derivatives In embodiments of the present invention, sarnath toxin linker derivatives or analogues may be utilized, comprising at least one non-natural amino acid or modified non-natural amino acid having a carbonyl, dicarbonyl, oxime, or hydroxylamine group. Methods for selecting and designing sarnath toxin linker derivatives to be modified using the methods, compositions, and techniques are well known in the art, see, for example, WO2013 / 185117, the entire contents of which are incorporated herein by reference. Sarnath toxin linker derivatives may be redesigned, including, by way of example only, as part of a high-throughput screening process (in which case many peptides may be designed, synthesized, characterized, and / or tested) or based on the researcher's interest. New sarnath toxin linker derivatives may also be designed based on the structure of known or partially characterized peptides. For example, the principles for selecting amino acids to be substituted and / or modified, and for selecting modifications to be used, are described in WO2013 / 185117. Sarnath toxin linker derivatives may be designed to meet the needs of experimenters or end users. Such needs may include, but are not limited to, manipulating the therapeutic efficacy of peptides, improving the safety properties of peptides, and modulating the pharmacokinetics, pharmacology, and / or pharmacodynamics of peptides, such as (by way of example only) increasing water solubility, bioavailability, prolonging serum half-life, prolonging therapeutic half-life, modulating immunogenicity, modulating biological activity, or prolonging circulation time. Furthermore, by way of example only, such modifications include providing additional functionality to the peptide, incorporating antibodies, and any combination of the foregoing modifications. Such saurus toxin linker derivatives may be modified to contain oxime, carbonyl, dicarbonyl, or hydroxylamine groups. Saurus toxin linker derivatives may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten or more carbonyl or dicarbonyl groups, oxime groups, hydroxylamine groups, or their protected forms. Sea haretoxin linker derivatives may be the same or different. For example, the derivatives may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different sites, which contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different reactive groups.

[0130] For example, sea haretoxin derivatives with a hydroxylamine (also known as an aminooxy) group as a linker allow for reaction with a variety of electrophilic groups to form conjugates, including but not limited to conjugates with PEG or other water-soluble polymers. Like hydrazides, acylhydrazides, and aminoureas, the enhanced nucleophilicity of the aminooxy group allows for efficient and selective reaction with a variety of molecules containing carbonyl or dicarbonyl groups, including but not limited to ketones, aldehydes, or other functional groups with similar chemical reactivity. (See, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899, 1995; H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9): 727-736, 2001). Although the reaction with the hydrazine group results in a corresponding hydrazone, oximes are generally produced by the reaction of the aminooxy group with carbonyl or dicarbonyl groups (such as, by way of example, ketones, aldehydes, or other functional groups with similar chemical reactivity). In some embodiments, sea haretoxin derivatives having a linker comprising an azide, alkyne, or cycloalkyne allow molecules to be linked via cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azide-alkyne Huisgen cycloaddition, etc., described in U.S. Patent 7,807,619, which is incorporated herein by reference to the extent of the reaction).

[0131] Therefore, in some embodiments, this document describes sea haretoxin derivatives having a linker comprising hydroxylamine, amino-oxygen, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketone-amine, ketone-alkyne, and alkene-diketone hydroxylamine groups, hydroxylamine-like groups (which have similar reactivity and structural similarity to hydroxylamine groups), masked hydroxylamine groups (which can be readily converted to hydroxylamine groups), or protected hydroxylamine groups (which have similar reactivity to hydroxylamine groups after deprotection). In some embodiments, sea haretoxin derivatives having a linker comprise azides, alkynes, or cycloalkynes. Examples of such sea haretoxin linker derivatives are also included elsewhere herein and in WO2013 / 185117 and WO2005 / 074650 (each incorporated herein by reference in its entirety).

[0132] Non-natural amino acids The selection of non-naturally encoded amino acid sites is based on surface exposure / site accessibility within the antibody, and hydrophobic or neutral amino acid sites are selected to maintain the charge on the antibody. Methods for inserting non-natural amino acids into sites in proteins are described, for example, in WO2010 / 011735 and WO2005 / 074650. This invention employs such methods and techniques. The non-natural amino acids used in the methods and compositions described herein have at least one of the following four characteristics: (1) at least one functional group on the side chain of the non-natural amino acid has at least one characteristic and / or activity and / or reactivity orthogonal to the chemical reactivity of 20 commonly encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) or at least one characteristic and / or activity and / or reactivity orthogonal to the chemical reactivity of naturally occurring amino acids present in polypeptides containing non-natural amino acids; (2) the introduced non-natural amino acid has a positive chemical reactivity with... The 20 commonly encoded amino acids are substantially chemically inert; (3) the non-natural amino acids can be stably incorporated into the polypeptide, preferably having stability comparable to that of naturally occurring amino acids or stability under typical physiological conditions, and more preferably such incorporation can occur via an in vivo system; and (4) the non-natural amino acids include oxime functional groups or functional groups that can be converted into oxime groups by reaction with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide including the non-natural amino acid (unless, of course, such destruction of biological properties is for the purpose of modification / conversion), or wherein the conversion can occur in aqueous conditions at a pH of about 4 to about 8, or wherein the reactive site on the non-natural amino acid is an electrophilic site. Any number of non-natural amino acids can be introduced into the polypeptide. Non-natural amino acids may also include protected or masked oximes or protected or masked groups that can be converted into oxime groups after the protected group is deprotected or the masked group is demasked. Non-natural amino acids may also include protected or masked carbonyl or dicarbonyl groups, which can be converted into carbonyl or dicarbonyl groups upon deprotection of the protected group or demasking of the masked group, thus allowing them to react with hydroxylamine or oxime to form oxime groups. Oxime-based non-natural amino acids can be synthesized by methods well known in the art (see, for example, WO2013 / 185117 and WO2005 / 074650), comprising: (a) reacting a hydroxylamine-containing non-natural amino acid with a carbonyl or dicarbonyl-containing reagent; (b) reacting a carbonyl or dicarbonyl-containing non-natural amino acid with a hydroxylamine-containing reagent; or (c) reacting an oxime-containing non-natural amino acid with certain carbonyl or dicarbonyl-containing reagents.

[0133] Non-natural amino acids that may be used in the methods and compositions described herein include, but are not limited to, amino acids containing amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, glycosylated amino acids (such as sugar-substituted serine), amino acids modified with other carbohydrates, ketone-containing amino acids, aldehyde-containing amino acids, amino acids containing polyethylene glycol or other polyethers, heavily atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids with extended side chains compared to natural amino acids (including, but not limited to, polyethers or long-chain hydrocarbons, including but not limited to, those with more than about 5 or more than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino acids containing aminothioic acids, and amino acids containing one or more toxic moieties.

[0134] In some embodiments, this document discloses anti-PSMA antibodies comprising one or more non-natural amino acids. The one or more non-natural amino acids may be encoded by a codon that does not encode one of the twenty natural amino acids. The one or more non-natural amino acids may be encoded by a nonsense codon (stop codon). The stop codon may be an amber codon. The amber codon may contain a UAG sequence. The stop codon may be an ochre codon. The ochre codon may contain a UAA sequence. The stop codon may be an opal or amber codon. The opal or amber codon may contain a UGA sequence. The one or more non-natural amino acids may be encoded by a tetrabase codon.

[0135] The non-natural amino acids disclosed herein include, but are not limited to, 1) substituted phenylalanine and tyrosine analogs, such as 4-amino-L-phenylalanine, 4-acetyl-L-phenylalanine, 4-azido-L-phenylalanine, 4-nitro-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-isopropyl-L-phenylalanine, 3-nitro-L-tyrosine, O-methyl-L-tyrosine, and O-phosphotyrosine; 2) photocrosslinkable amino acids, such as amino acids having aryl azide or benzophenone groups, such as 4-azido-phenylalanine or 4-benzoylphenylalanine; 3) amino acids with unique chemical reactivity, such as 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-2-propynyl-1-yl-L-tyrosine, N-(ethio)thiocarbonyl- 4) L-phenylalanine and p-(3-oxobutyryl)-L-phenylalanine; 5) amino acids containing heavy atoms, such as 4-iodo-L-phenylalanine or 4-bromo-L-phenylalanine used for phase determination in X-ray crystallography; 6) redox-active amino acids, such as 3,4-dihydroxy-L-phenylalanine; 7) fluorinated amino acids, such as 2-fluorophenylalanine (e.g., 2-fluoro-L-phenylalanine), 3-fluorophenylalanine (e.g., 3-fluoro-L-phenylalanine), or 4-fluorophenylalanine (e.g., 4-fluoro-L-phenylalanine); 8) fluorescent amino acids, such as amino acids containing naphthyl, dansyl, or 7-aminocoumarin side chains; 9) photolytically cleavable or photoisomerizable amino acids, such as amino acids containing azobenzyl or nitrobenzyl groups, such as cysteine, serine, or tyrosine containing azobenzyl or nitrobenzyl groups; 10) β-amino acids (e.g., β-amino acids). 2 or β 310) Homoamino acids, such as high-glutamine (e.g., β-high-glutamine) or high-phenylalanine (e.g., β-high-phenylalanine); 11) Proline or pyruvate derivatives; 12) 3-substituted alanine derivatives; 14) Glycine derivatives; 15) Linear core amino acids; 16) Diamino acids; 17) D-amino acids; 18) N-methyl amino acids; 19) Phosphotyrosine mimics, such as carboxymethylphenylalanine (pCmF) (e.g., 4-carboxymethyl-L-phenylalanine); 20) 2-Aminooctanoic acid; and 21) amino acids containing a sugar moiety, such as N-acetyl-L-glucosamine-L-serine, β-N-acetylglucosamine-O-serine, N-acetyl-L-galactosamine-L-serine, α-N-acetylgalactosamine-O-serine, O-(3-OD-galactosyl-N-acetyl-β-D-galactosamine)-L-serine, N-acetyl-L-glucosamine-L-threonine, α-N-acetylgalactosamine-O-threonine, 3-O-(N-acetyl-L-glucosamine-L-threonine)-serine, α-N-acetylgalactosamine-O-threonine, 3-O-(N-acetyl-L-glucosamine-L-threonine)-serine, α-N-acetylgalactosamine-O-threonine, α-O-(N-acetyl-L-glucosamine-L-threonine)-serine, β-N-acetyl-glucosamine-O-serine, β-N-acetyl-glucosamine-O-serine, β-N-acetyl-glucosamine-O-serine, β-N-acetyl-L-glucosamine-L-threon ... -β-D-glucosinolate)-L-threonine, N-acetyl-L-glucosinolate-L-asparagine, N4-(β-N-acetyl-D-glucosinolate)-L-asparagine and O-(mannosyl)-L-serine; wherein the naturally occurring N- or O- bonds between the amino acid and the sugar are replaced by covalent bonds (including but not limited to alkenes, oximes, thioethers, amides, etc.) that are not common in nature; or amino acids containing sugars (such as 2-deoxyglucose, 2-deoxygalactose, etc.) that are not common in naturally occurring polypeptides.Specific examples of non-natural amino acids include, but are not limited to, p-acetylphenylalanine (4-acetylphenylalanine) (including 4-acetyl-L-phenylalanine, also referred to herein as p-acetyl-L-phenylalanine (pAF)), 4-boronylphenylalanine (pBoF) (e.g., 4-boron-L-phenylalanine), 4-propynyloxyphenylalanine (pPrF) (e.g., 4-propynyloxy-L-phenylalanine), and O-methyltyrosine (e.g., O-methyl-L-tyrosine). Acids), 3-(2-naphthyl)alanine (NapA) (e.g., 3-(2-naphthyl)-L-alanine), 3-methylphenylalanine (e.g., 3-methyl-L-phenylalanine), O-allyltyrosine (e.g., O-allyl-L-tyrosine), O-isopropyltyrosine (e.g., O-isopropyl-L-tyrosine), dopamine (e.g., L-DOPA), 4-isopropylphenylalanine (e.g., 4-isopropyl-L-phenylalanine), 4-azidophenylalanine Acids (pAz) (e.g., 4-azido-L-phenylalanine), 4-benzoyl-L-phenylalanine (pBpF) (e.g., 4-benzoyl-L-phenylalanine), O-phosphoserine (e.g., O-phospho-L-serine), O-phosphotyrosine (e.g., O-phospho-L-tyrosine), 4-iodophenylalanine (pIF) (e.g., 4-iodo-L-phenylalanine, 4-bromophenylalanine, 4-bromo-L-phenylalanine, 4-aminophenylalanine) (e.g., 4-amino-L-phenylalanine), 4-cyanophenylalanine (pCNF) (e.g., 4-cyano-L-phenylalanine), (8-hydroxyquinoline-3-yl)alanine (HQA) (e.g., (8-hydroxyquinoline-3-yl)-L-alanine), (2,2-bipyridin-5-yl)alanine (BipyA) (e.g., (2,2-bipyridin-5-yl)-L-alanine), etc. Other non-natural amino acids are disclosed in Liu et al. (2010). Annu Rev Biochem, 79:413-44; Wang et al. (2005) Angew Chem IntEd, 44:34-66; and published international applications: WO 2012 / 166560, WO 2012 / 166559, WO 2011 / 028195, WO2010 / 037062, WO 2008 / 083346, WO 2008 / 077079, WO 2007 / 094916, WO 2007 / 079130, WO2007 / 070659 and WO 2007 / 059312, the entire contents of each of which are hereby incorporated herein by reference. In some embodiments, one or more non-natural amino acids may be p-acetylphenylalanine. In some more specific embodiments, one or more non-natural amino acids may be p-acetyl-L-phenylalanine (pAF).

[0136] In some embodiments, one or more non-natural amino acids are selected from the group consisting of: 4-acetylphenylalanine, 3-O-(N-acetyl-β-D-glucosamine)threonine, N4-(β-N-acetyl-D-glucosamine)asparagine, O-allyltyrosine, α-N-acetylgalactosamine-O-serine, α-N-acetylgalactosamine-O-threonine, 2-aminooctanoic acid, 2-aminophenylalanine, 3-aminophenylalanine 4-Aminophenylalanine, 2-Aminotyrosine, 3-Aminotyrosine, 4-Azide-phenylalanine, 4-Benzoylphenylalanine, (2,2-Bipyridin-5-yl)alanine, 3-Boronphenylalanine, 4-Boronphenylalanine, 4-Bromophenylalanine, p-Carboxymethylphenylalanine, 4-Carboxyphenylalanine, p-Cyanophenylalanine, 3,4-Dihydroxyphenylalanine, 4-Ethynylphenylalanine, 2-Fluorophenylalanine, 3-Fluorophenylalanine, 4-Fluorophenylalanine, O -(3-OD-galactosyl-N-acetyl-β-D-galactosamine)serine, high-glutamine, (8-hydroxyquinoline-3-yl)alanine, 4-iodophenylalanine, 4-isopropylphenylalanine, O-isopropyltyrosine, 3-isopropyltyrosine, O-mannopyranosylserine, 2-methoxyphenylalanine, 3-methoxyphenylalanine, 4-methoxyphenylalanine, 3-methylphenylalanine, O-methyltyrosine, 3-(2-naphthyl)alanine 5-Nitrohistidine, 4-Nitrohistidine, 4-Nitroleucine, 2-Nitrophenylalanine, 3-Nitrophenylalanine, 4-Nitrophenylalanine, 4-Nitrotryptophan, 5-Nitrotryptophan, 6-Nitrotryptophan, 7-Nitrotryptophan, 2-Nitrotyrosine, 3-Nitrotyrosine, O-phosphoserine, O-phosphotyrosine, 4-propynyloxyphenylalanine, O-2-propynyl-1-yltyrosine, 4-sulfophenylalanine, and O-sulfotyrosine.

[0137] In some alternative embodiments, one or more non-natural amino acids are selected from the group consisting of: 4-acetyl-L-phenylalanine (p-acetyl-L-phenylalanine (pAF)), 3-O-(N-acetyl-β-D-glucosamine)-L-threonine, N4-(β-N-acetyl-D-glucosamine)-L-asparagine, O-allyl-L-tyrosine, α-N-acetylgalactosamine-OL-serine, α-N-acetylgalactosamine-OL-threonine, 2-aminooctanoic acid, 2-amino-L-phenylalanine, 3-amino-L-phenylalanine, 4-amino-L-phenylalanine, and so on. -Phenylalanine, 2-amino-L-tyrosine, 3-amino-L-tyrosine, 4-azido-L-phenylalanine, 4-benzoyl-L-phenylalanine, (2,2-bipyridin-5-yl)-L-alanine, 3-boron-L-phenylalanine, 4-boron-L-phenylalanine, 4-bromo-L-phenylalanine, p-carboxymethyl-L-phenylalanine, 4-carboxy-L-phenylalanine, p-cyano-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine (L-DOPA), 4-ethynyl-L-phenylalanine, 2-fluoro-L-phenylalanine, 3-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine Acids, O-(3-OD-galactosyl-N-acetyl-β-D-galactosamine)-L-serine, L-high glutamine, (8-hydroxyquinoline-3-yl)-L-alanine, 4-iodo-L-phenylalanine, 4-isopropyl-L-phenylalanine, O-isopropyl-L-tyrosine, 3-isopropyl-L-tyrosine, O-mannopyranosyl-L-serine, 2-methoxy-L-phenylalanine, 3-methoxy-L-phenylalanine, 4-methoxy-L-phenylalanine, 3-methyl-L-phenylalanine, O-methyl-L-tyrosine, 3-(2-naphthyl)-L-alanine, 5-nitro The non-natural amino acids are p-L-histidine, 4-nitro-L-histidine, 4-nitro-L-leucine, 2-nitro-L-phenylalanine, 3-nitro-L-phenylalanine, 4-nitro-L-phenylalanine, 4-nitro-L-tryptophan, 5-nitro-L-tryptophan, 6-nitro-L-tryptophan, 7-nitro-L-tryptophan, 2-nitro-L-tyrosine, 3-nitro-L-tyrosine, O-phospho-L-serine, O-phospho-L-tyrosine, 4-propynyloxy-L-phenylalanine, O-2-propynyl-1-yl-L-tyrosine, 4-sulfo-L-phenylalanine, and O-sulfo-L-tyrosine. In some embodiments, one or more non-natural amino acids may be p-acetyl-L-phenylalanine (pAF). Therefore, in some embodiments, each of the one or more non-natural amino acids is a pAF.

[0138] In certain embodiments of this disclosure, antibodies having at least one non-natural amino acid include at least one post-translational modification. In one embodiment, the at least one post-translational modification comprises attaching a molecule containing a second reactive group (including, but not limited to, water-soluble polymers, polyethylene glycol derivatives, pharmaceuticals, second proteins or peptides or peptide analogs, antibodies or antibody fragments, bioactive agents, small molecules, or any combination of the foregoing or any other desired compound or substance) to at least one non-natural amino acid containing a first reactive group using chemical methods known to those skilled in the art for a particular reactive group. For example, the first reactive group is an alkynyl moiety (including, but not limited to, the non-natural amino acid p-propynyloxyphenylalanine, wherein the propynyl group is sometimes also referred to as the acetylene moiety), and the second reactive group is an azide moiety, and a [3+2] cycloaddition chemical method is used. In another example, the first reactive group is an azide moiety (including, but not limited to, the non-natural amino acid p-azido-L-phenylalanine), and the second reactive group is an alkynyl moiety. In some embodiments of the modified antibody peptides disclosed herein, at least one non-natural amino acid (including, but not limited to, non-natural amino acids containing a ketone functional group) comprising at least one post-translational modification is used, wherein the at least one post-translational modification comprises a sugar moiety. In some embodiments, the post-translational modification is performed in vivo in eukaryotic or non-eukaryotic cells. In other embodiments, the post-translational modification is performed in vitro. In yet another embodiment, the post-translational modification is performed both in vitro and in vivo.

[0139] In some embodiments, the non-natural amino acid may be modified to incorporate a chemical group. In some embodiments, the non-natural amino acid may be modified to incorporate a ketone group. One or more non-natural amino acids may contain at least one oxime, carbonyl, dicarbonyl, hydroxylamine group, or a combination thereof. One or more non-natural amino acids may contain at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic olefin, acyclic alkyne, cyclooctyne, aryl / alkyl azide, norbornene, cyclopropene, trans-cyclooctene, or tetrazine functional group, or a combination thereof.

[0140] In some embodiments disclosed herein, non-natural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. Methods for incorporating non-natural amino acids into molecules (e.g., proteins, polypeptides, or peptides) are disclosed in U.S. Patents: 7,332,571; 7,928,163; 7,696,312; 8,008,456; 8,048,988; 8,809,511; 8,859,802; 8,791,231; 8,476,411; or 9,637,411 (each of which is incorporated herein by reference in its entirety) and the examples herein. One or more non-natural amino acids can be incorporated by methods known in the art. For example, cell-based or cell-free systems can be used, and auxotrophic strains can be used instead of engineered tRNA and synthases. In some embodiments, such as those disclosed in, for example, WO2002085923A2; WO2002086075A2; WO2004035743A2; WO2007021297A1; WO2006068802A2; and WO2006069246A2, an orthogonal tRNA synthetase is used, the contents of each of which are incorporated herein by reference in their entirety. Incorporating one or more non-natural amino acids into an antibody or antibody fragment or variant may include modifying one or more amino acid residues in the antibody or antibody fragment or variant. Modifying one or more amino acid residues in the antibody or antibody fragment or variant may include mutating one or more nucleotides in the nucleotide sequence encoding the antibody or antibody fragment or variant. Mutating one or more nucleotides in the nucleotide sequence encoding the antibody or antibody fragment or variant may include changing the codon encoding the amino acid to a nonsense codon. Incorporating one or more non-natural amino acids into an antibody or antibody fragment or variant may include modifying one or more amino acid residues in the antibody or antibody fragment or variant to produce one or more amber codons in the antibody or antibody fragment or variant. One or more non-natural amino acids may be incorporated into antibodies, antibody fragments, or variants in response to amber codons. One or more non-natural amino acids may be incorporated into antibodies, antibody fragments, or variants site-specifically. Incorporation of one or more non-natural amino acids into antibodies, antibody fragments, or variants may comprise one or more genetically encoded non-natural amino acids that exhibit orthogonal chemical reactivity relative to the typical twenty amino acids to site-specifically modify bioactive molecules or targets. Incorporation of one or more non-natural amino acids may include the use of tRNA / aminoacyl-tRNA synthetase pairs to site-specifically incorporate one or more non-natural amino acids at defined sites in bioactive molecules or targets in response to one or more amber nonsense codons.Other methods for incorporating non-natural amino acids include, but are not limited to, those disclosed in Chatterjee et al., A Versatile Platform for Single- and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli, Biochemistry, 2013; Kazane et al., J Am Chem Soc, 135(1):340-6, 2013; Kim et al., J Am Chem Soc, 134(24):9918-21, 2012; Johnson et al., Nat Chem Biol, 7(11):779-86, 2011; and Hutchins et al., J Mol Biol, 406(4):595-603, 2011. One or more non-natural amino acids can be produced by a selective reaction of one or more natural amino acids. The selective reaction can be mediated by one or more enzymes. In a non-limiting example, the selective reaction of one or more cysteine ​​residues with formylglycine synthase (FGE) can produce one or more formylglycine residues, as described in Rabuka et al., Nature Protocols 7: 1052-1067, 2012. One or more non-natural amino acids may be involved in chemical reactions for linker formation. Chemical reactions for linker formation may include bioorthogonal reactions. Chemical reactions for linker formation may include click chemistry. See, for example, WO2006 / 050262, which is incorporated herein by reference in its entirety.

[0141] In some embodiments, the non-natural amino acid comprises a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-asparagine, and O-mannosamine-L-serine. Examples of such amino acids also include those where the naturally occurring N- or O-bond between the amino acid and the sugar is replaced by a covalent bond (including, but not limited to, alkenes, oximes, thioethers, amides, etc.) that is not commonly found in nature. Examples of such amino acids also include sugars (such as 2-deoxyglucose, 2-deoxygalactose, etc.) that are not commonly found in naturally occurring proteins.

[0142] The incorporation of chemical moieties into peptides via the incorporation of non-natural amino acids offers numerous advantages and manipulation benefits. For example, the unique reactivity of carbonyl or dicarbonyl functional groups (including ketone or aldehyde functional groups) allows for selective modification of proteins in vivo and in vitro using either hydrazine- or hydroxylamine-containing reagents. For instance, heavy-atom non-natural amino acids can be used for phase-determining X-ray structural data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in selecting the position of heavy atoms. Photoreactive non-natural amino acids (including, but not limited to, amino acids with benzophenone and aryl azides, including, but not limited to, phenyl azide side chains) allow for efficient in vivo and in vitro photocrosslinking of peptides, for example. Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine. Peptides with photoreactive non-natural amino acids can then be arbitrarily crosslinked by exciting photoreactive groups, providing temporal control. In a non-limiting example, the methyl groups of non-natural amino acids can be substituted with isotopically labeled (including, but not limited to) methyl groups as probes of local structure and dynamics, including, but not limited to, the use of nuclear magnetic resonance and vibrational spectroscopy.

[0143] Non-natural amino acid-linked sea haretoxin derivatives In other embodiments of the invention, methods, strategies, and techniques for incorporating at least one sea slug toxin linker derivative into a non-natural amino acid are described herein. The invention described herein includes methods for producing, purifying, characterizing, and using sea slug toxin linker derivatives containing at least one such non-natural amino acid. This aspect also includes compositions (including DNA and RNA) that can be used to at least partially produce sea slug toxin linker derivatives containing at least one non-natural amino acid, and methods for producing, purifying, characterizing, and using oligonucleotides. This aspect also includes compositions capable of expressing such oligonucleotides that can at least partially produce sea slug toxin linker derivatives containing at least one non-natural amino acid, and methods for producing, purifying, characterizing, and using these cells.

[0144] Therefore, this document provides and describes sea haretoxin linker derivatives comprising at least one non-natural amino acid or a modified non-natural amino acid having a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime, or hydroxylamine group. In some embodiments, the sea haretoxin linker derivative having at least one non-natural amino acid or a modified non-natural amino acid having a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime, or hydroxylamine group includes at least one post-translational modification at a position on the polypeptide. In some embodiments, co-translation or post-translational modification occurs via cellular mechanisms (e.g., glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid bond modification, etc.). In many cases, such cellular mechanism-based co-translation or post-translational modification occurs at naturally occurring amino acid sites on the polypeptide; however, in some embodiments, cellular mechanism-based co-translation or post-translational modification occurs at non-natural amino acid sites on the polypeptide.

[0145] In other embodiments, post-translational modification does not utilize cellular mechanisms but instead provides functionality through the attachment of a molecule (polymer; water-soluble polymer; polyethylene glycol derivative; second protein or polypeptide or polypeptide analog; antibody or antibody fragment; and any combination thereof) containing a second reactive group to at least one non-natural amino acid containing a first reactive group (including, but not limited to, a non-natural amino acid containing a ketone, aldehyde, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine functional group). In some embodiments, co-translation or post-translational modification is performed in vivo in eukaryotic or non-eukaryotic cells. In some embodiments, post-translational modification is performed in vitro and does not utilize cellular mechanisms. This aspect also includes methods for producing, purifying, characterizing, and using such sea haretoxin linker derivatives containing at least one such co-translated or post-translational modified non-natural amino acid.

[0146] Within the scope of the methods, compositions, strategies, and techniques described herein, therein also includes reagents capable of reacting with a sea haretoxin linker derivative (containing a carbonyl or dicarbonyl group, an oxime group, an alkyne, a cycloalkyne, an azide, a hydroxylamine group, or a masked or protected form thereof) that is part of a polypeptide to produce any of the aforementioned post-translational modifications. In some embodiments, the resulting post-translational modified sea haretoxin linker derivative will contain at least one oxime group; the resulting modified oxime-containing sea haretoxin linker derivative is susceptible to subsequent modification reactions. This aspect also includes methods for producing, purifying, characterizing, and using such reagents capable of performing any such post-translational modifications on such sea haretoxin linker derivatives.

[0147] In some embodiments, the peptide or non-naturally amino acid-linked saurus toxin derivative includes at least one co-translational or post-translational modification performed in vivo by one host cell type, wherein the post-translational modification is generally not performed by another host cell type. In some embodiments, the peptide includes at least one co-translational or post-translational modification performed in vivo by eukaryotic cells, wherein the co-translational or post-translational modification is generally not performed by non-eukaryotic cells. Examples of such co-translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, phosphorylation, glycolipid bond modification, etc. In one embodiment, the co-translational or post-translational modification includes attaching an oligosaccharide to asparagine via a GlcNAc-asparagine bond (including, but not limited to, oligosaccharides including (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, etc.). In another embodiment, co-translation or post-translational modification includes attaching oligosaccharides (including, but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to serine or threonine residues via GalNAc-serine, GalNAc-threonine, GlcNAc-serine, or GlcNAc-threonine bonds. In some embodiments, the protein or peptide may contain secreted or localizing sequences, epitope tags, FLAG tags, polyhistidine tags, GST fusions, etc. This aspect also includes methods for producing, purifying, characterizing, and using such peptides containing at least one of these co-translational or post-translational modifications. In other embodiments, the glycosylated non-natural amino acid peptides are generated in a non-glycosylated form. This non-glycosylated form of glycosylated non-natural amino acids can be produced by a method comprising the following steps: chemically or enzymatically removing oligosaccharide groups from isolated, substantially purified, or unpurified glycosylated non-natural amino acid peptides; producing the non-natural amino acid in a host that does not glycosylate the non-natural amino acid peptide (such host includes prokaryotes or eukaryotes engineered or mutated to not glycosylate the peptide); introducing a glycosylation inhibitor into a cell culture medium in which the non-natural amino acid peptide is produced by eukaryotes that would normally glycosylate the peptide; or any combination of such methods. This document also describes this non-glycosylated form of normally glycosylated non-natural amino acid peptides (normal glycosylation refers to a peptide that would be glycosylated when produced under conditions where naturally occurring peptides are glycosylated). Of course, this non-glycosylated form of normally glycosylated non-natural amino acid peptides (or indeed any peptide described herein) can be in an unpurified, substantially purified, or isolated form.

[0148] Oxime-linked sea haretoxin derivatives Derivatives of non-natural amino acids linked to oxime groups via sea haretoxin can be reacted with various reagents containing certain reactive carbonyl or dicarbonyl groups (including, but not limited to, ketones, aldehydes, or other groups with similar reactivity) to form new non-natural amino acids containing novel oxime groups. Such oxime exchange reactions allow for further functionalization of sea haretoxin-linked derivatives. Furthermore, the original sea haretoxin-linked derivatives containing oxime groups can be used independently, provided that the oxime bond is stable under the conditions necessary for incorporating the amino acid into the polypeptide (e.g., in vivo, in vitro, and chemical synthesis methods described herein, as well as in WO2013 / 185117 and WO2005 / 074650, each of which is incorporated herein by reference).

[0149] Therefore, in some embodiments, this document describes derivatives having non-natural amino acid samosatoxin linked to side chains comprising an oxime group, an oxime-like group (which has similar reactivity to an oxime group and is structurally similar to an oxime group), a masked oxime group (which can be readily converted into an oxime group), or a protected oxime group (which has similar reactivity to an oxime group after deprotection).

[0150] Methods and compositions for incorporating one or more non-natural amino acids into sea haretoxin linker derivatives are well known in the art (see, for example, WO2013 / 185117 and WO2005 / 074650, each incorporated herein by reference in its entirety). One or more non-natural amino acids may be incorporated into one or more specific sites without disrupting the activity of the sea haretoxin linker derivative. This can be achieved by performing “conservative” substitutions, including but not limited to substituting hydrophobic amino acids with non-natural or natural hydrophobic amino acids, substituting bulky amino acids with non-natural or natural bulky amino acids, substituting hydrophilic amino acids with non-natural or natural hydrophilic amino acids, and / or inserting non-natural amino acids into positions where activity is not desired.

[0151] Various biochemical and structural methods can be employed to select desired sites within the sea slug toxin linker derivative for substitution with non-natural amino acids. In some embodiments, the non-natural amino acid is linked to the C-terminus of the sea slug toxin derivative. In other embodiments, the non-natural amino acid is linked to the N-terminus of the sea slug toxin derivative. Any position within the sea slug toxin linker derivative is suitable for selection to incorporate a non-natural amino acid, and selection can be based on rational design or by random selection for any or no specific desired purpose. The selection of desired sites can be based on generating a non-natural amino acid polypeptide (which may be further modified or remain unmodified) with any desired property or activity, including but not limited to receptor binding modulators, receptor activity modulators, modulators of binding to binding couplers, binding coupler activity modulators, binding coupler conformation modulators, dimer or multimer formation, no change in activity or property compared to the natural molecule, or manipulation of any physical or chemical property of the polypeptide such as solubility, aggregation, or stability. Alternatively, sites identified as essential to biological activity can also be good candidates for substitution with non-natural amino acids, again depending on the desired activity sought for the polypeptide. Another alternative is to simply substitute non-natural amino acids sequentially at each position on the polypeptide chain and observe the effect on the polypeptide's activity. Any means, techniques, or methods used to select the positions in any polypeptide to be substituted with non-natural amino acids are applicable to the methods, techniques, and compositions described herein.

[0152] The structure and activity of naturally occurring mutants containing the missing peptide can also be examined to identify protein regions that may be resistant to non-natural amino acid substitutions. Once residues that may be intolerant to non-natural amino acid substitutions have been eliminated, the effects of the proposed substitutions at each of the remaining positions can be examined using methods including, but not limited to, the three-dimensional structure of the relevant peptide and any relevant ligands or binding proteins. X-ray crystallography and NMR structures of many peptides are available in the Protein Database (PDB, see the World Wide Web at rcsb.org), a centralized database of three-dimensional structural data for macromolecules such as proteins and nucleic acids, which can be used to identify amino acid positions that can be substituted by non-natural amino acids. Furthermore, if three-dimensional structural data are not available, models of the secondary and tertiary structures of the peptide can be created to study them. Thus, the identity of amino acid positions that can be substituted by non-natural amino acids can be readily obtained.

[0153] Exemplary sites for incorporating non-natural amino acids include, but are not limited to, those excluded from potential receptor-binding regions, or regions that bind to binding proteins or ligands that are fully or partially exposed to solvents, have minimal or no hydrogen bond interactions with nearby residues, are minimally exposed to nearby reactive residues, and / or are located in highly flexible regions as predicted by the three-dimensional crystal structure of a particular polypeptide and its associated receptor, ligand, or binding protein.

[0154] Various non-natural amino acids can be substituted or incorporated into a given position in a polypeptide. As an example, specific non-natural amino acids for incorporation can be selected based on examination of the three-dimensional crystal structure of the polypeptide and its associated ligands, receptors, and / or binding proteins, with conservative substitution being preferred.

[0155] In one embodiment, the method described herein includes: incorporating a sea haretoxin linker derivative, wherein the sea haretoxin linker derivative comprises a first reactive group; and contacting the sea haretoxin linker derivative with a molecule comprising a second reactive group (including, but not limited to, a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof). In some embodiments, the first reactive group is a hydroxylamine moiety, and the second reactive group is a carbonyl or dicarbonyl moiety, thereby forming an oxime bond. In some embodiments, the first reactive group is a carbonyl or dicarbonyl moiety, and the second reactive group is a hydroxylamine moiety, thereby forming an oxime bond. In some embodiments, the first reactive group is a carbonyl or dicarbonyl moiety, and the second reactive group is an oxime moiety, thereby undergoing an oxime exchange reaction. In some embodiments, the first reactive group is an oxime moiety, and the second reactive group is a carbonyl or dicarbonyl moiety, thereby undergoing an oxime exchange reaction.

[0156] In some cases, the incorporation of the sea haretoxin linker derivative will combine with other additions, substitutions, or deletions within the peptide to affect other chemical, physical, pharmacological, and / or biological properties. In some cases, other additions, substitutions, or deletions may increase the stability of the peptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the peptide for its appropriate receptors, ligands, and / or binding proteins. In some cases, other additions, substitutions, or deletions may increase the solubility of the peptide (including, but not limited to, when expressed in *E. coli* or other host cells). In some embodiments, to increase the solubility of the peptide after expression in *E. coli* or other recombinant host cells, sites selected for incorporation with naturally encoded or non-natural amino acids are chosen, in addition to another site for incorporation of non-natural amino acids. In some embodiments, the peptide contains another addition, substitution, or deletion that modulates affinity for the relevant ligand, binding protein, and / or receptor, modulates (including, but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bioavailability, promotes purification, or improves or alters a particular route of administration. Similarly, non-natural amino acid peptides may contain chemically or enzymatically cleaved sequences, protease cleaved sequences, reactive groups, antibody-binding domains (including but not limited to FLAG or poly-His) or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification, transport across tissue or cell membranes, prodrug release or activation, size reduction, or other properties of the peptide.

[0157] In some embodiments, the payload or toxin portion is incorporated into the anti-PSMA antibody-drug conjugate disclosed herein. Examples of payloads may include, in a non-limiting manner, DNA replication inhibitors, DNA transcription inhibitors, RNA translation inhibitors, cell division inhibitors, cell signaling inhibitors, kinase inhibitors, tubulin polymerase inhibitors, tubulin depolymerizing agents, DNA cleaving agents, DNA binding agents, RNA polymerase inhibitors, oliquistatin, salivarius toxin, MMAF, MMAE, MMAD, pyrrolizin analogues, pyrrolobenzodiazepine (PBD) analogues, tubulolysin analogues, maytansin analogues, muscarinic acid analogues, novozygosin analogues, epothilone analogues, chachiin analogues, doxorubicin analogues, and camptothecin analogues.

[0158] In other embodiments of the invention, drug payload linkers are provided. Anti-microtubule inhibitors are selected as payloads in combination with cleavable, short cleavable, and non-cleavable linkers. In one exemplary manner, the payload linker combinations disclosed herein may include, but are not limited to, cleavable, non-cleavable, and short cleavable payload linkers. Cleavable payload linkers may include cleavable dipeptides (including but not limited to Val-Cti, Val-Ala, Val-Lys, and Ala-Ala), hydrazine bonds, disulfide bonds, or pyrophosphate bonds. Examples of payload linkers used in the anti-PSMA antibody drug conjugates of this disclosure include non-cleavable MMAE, non-cleavable MMAF, Val-citrulline-acetyl MMAF, short Val-citrulline-acetyl MMAF, or short Val-citrulline-acetyl MMAE. Both MMAE and MMAF are used in studies. Non-limiting examples of sarsaparilla toxin linker derivatives include the following: Non-degradable MMAF (AS269) with the following structure: .

[0159] Non-decomposable MMAEs with the following structures: .

[0160] Cleavable or Val-citrulline-acetyl (Val-Cit)MMAF with the following structure: .

[0161] Short cleavable or short Val-citrulline-acetyl (Val-Cit)MMAFs with the following structure: .

[0162] Short, cleavable or short Val-citrulline-acetyl (Val-Cit)MMAEs with the following structure: .

[0163] Tables 1 and 2 provide drug-connector compounds that can be used with the anti-PSMA antibodies or antibody-drug conjugates of the present invention. The synthesis of such payload connectors is well known to those skilled in the art. See, for example, Dubowchik et al., Bioconjugate Chem. 13: 855-869, (2002); Doronina et al., Nature Biotechnology 21(7): 778-784, (2003); WO2012 / 166560; WO2013 / 185117, the entire contents of each of which are incorporated herein by reference.

[0164] Table 1. Drug-Connector Compound-Single Payload Table 2. Drug-Connector Compound-Dual Loading In some aspects of this disclosure, it is desirable for anti-PSMA antibodies, variants, or pharmaceutical conjugates to have increased serum half-life, water solubility, bioavailability, therapeutic half-life, or cycling time, or to modulate immunogenicity or biological activity. One method to achieve such desired characteristics of the anti-PSMA compositions disclosed herein is through covalent linkage of a polymer, polyethylene glycol (PEG). To maximize the desired properties of the PEG, the total molecular weight and hydration state of one or more polymers attached to the bioactive molecule must be sufficiently high to impart the advantageous properties typically associated with such polymer attachment, such as increased water solubility and cycling half-life, without adversely affecting the biological activity of the molecule attached to the PEG.

[0165] Polyethylene glycol derivatives are typically linked to bioactive molecules via reactive chemical functional groups, such as amino acid residues, N-termini, and / or carbohydrate moieties. WO99 / 67291 discloses a method for conjugating proteins with polyethylene glycol, wherein at least one amino acid residue on the protein is replaced by a synthetic amino acid, and the protein is contacted with polyethylene glycol under conditions sufficient to achieve protein conjugation.

[0166] Proteins and other molecules typically have a limited number of reactive sites available for polymer attachment. Sites most suitable for modification via polymer attachment may play a crucial role in receptor binding, and such sites may be essential for preserving the molecule's biological activity, thus making them unsuitable for polymer attachment. Therefore, indiscriminate attachment of polymer chains to such reactive sites on bioactive molecules often results in a significant reduction or even complete loss of the bioactivity of the polymer-modified molecule. Polyethylene glycol (PEG) attachment can target specific sites within a protein, ensuring that the PEG moiety does not interfere with the protein's function. One method for guiding PEG attachment is to introduce synthetic amino acids into the protein sequence. The protein biosynthesis mechanism of the prokaryotic Escherichia coli (E. coli) can be altered to efficiently and faithfully incorporate synthetic amino acids into proteins in response to the amber codon UAG. See, for example, JW Chin et al., J. Amer. Chem. Soc. 124: 9026-9027, 2002; JW Chin, & PG Schultz, ChemBioChem 3(11): 1135-1137, 2002; JW Chin et al., PNAS USA 99: 11020-11024, 2002; and L. Wang, & PG Schultz, Chem. Comm., 1: 1-11, 2002. A similar method can be accomplished using the eukaryotic yeast Saccharomyces cerevisiae / S. cerevisiae (e.g., J. Chin et al., Science 301: 964-7, 2003). Using this method, non-naturally encoded amino acids can be incorporated into the anti-PSMA antibodies, variants, or pharmaceutical conjugates of the present invention, thereby providing attachment sites for polyethylene glycol. See, for example, WO2010 / 011735 and WO2005 / 074650.

[0167] Pharmaceutical Composition In other respects, this disclosure provides pharmaceutical compositions or formulations containing the anti-PSMA antibody, antibody fragment, variant, or anti-PSMAADC of this disclosure. Such pharmaceutical compositions may use a variety of pharmaceutically acceptable excipients, stabilizers, buffers, and other components for administration to animals. See, for example, Remington, *The Science and Practice of Pharmacy*, 19th edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995. Because multiple components need to be considered (e.g., purified, stabilizing components), the stability, administration to subjects, and activity of suitable compositions or formulations vary with each compound. Suitable salts included in the composition or formulation may include, but are not limited to, sodium chloride, potassium chloride, or calcium chloride. Buffers and / or stabilizers, such as sodium acetate, may be used. Suitable buffers may include, alone or in combination, acetate buffers, phosphate-citrate buffers, phosphate buffers, citrate buffers, histidine buffers, L-histidine, L-arginine hydrochloride, bicarbonate buffers, succinate buffers, citrate buffers, and TRIS buffers. Surfactants may also be used, including polysorbates (e.g., polysorbate 80), dodecyl sulfate (SDS), and lecithin, either alone or in combination.

[0168] In some embodiments, the pharmaceutical composition may be a formulation comprising the anti-PSMA ADC of this disclosure and one or more pharmaceutically acceptable excipients, stabilizers or buffers.

[0169] In some implementations, the anti-PSMA ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each of the heavy chains at position 114 according to the Kabat number, and wherein a drug linker is conjugated to each of the pAFs via an oxime bond, wherein each of the drug linkers is amberstatin269 (AS269) having the following structure: .

[0170] In some implementations, the anti-PSMA ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain contains a heavy chain variable region of SEQ ID NO: 1, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each said heavy chain at position 114 according to the Kabat number; wherein a drug-linker is conjugated to each said pAF via an oxime bond, wherein each said drug-linker is amberstatin269 (AS269) having the following structure: .

[0171] In some implementations, the anti-PSMA ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain contains the heavy chain variable region of SEQ ID NO:1, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, wherein each light chain contains the light chain variable region of SEQ ID NO:2; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0172] In some embodiments, the anti-PSMA ADC comprises a heavy chain having the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-PSMA ADC comprises a light chain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-PSMA ADC comprises a heavy chain having the amino acid sequence of SEQ ID NO: 8 and a light chain having the amino acid sequence of SEQ ID NO: 9.

[0173] In some implementations, the PSMA-resistant ADC is an ARX517. In some implementations, the PSMA-resistant ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO:8, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, and each light chain has the amino acid sequence of SEQ ID NO:9; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0174] In some aspects, the pharmaceutical composition or formulation may be in the form of an aqueous composition or a reconstituted liquid composition or a powder. When the formulation is in liquid form, the composition or formulation may have a pH range of about 4.0 to about 7.0 or about 4.5 to about 6.5. However, a skilled physician may adjust the pH to provide acceptable stability and administration. The composition may also be stored in vials or cartridges, pen delivery devices, syringes, intravenous administration tubing, or intravenous administration bags, but is not limited thereto.

[0175] The formulation selection criteria for the anti-PSMA ADC disclosed herein may include solubility, chemical stability, and physical stability.

[0176] In some embodiments, the formulation may contain the anti-PSMA ADC and buffer, sugar or surfactant of this disclosure; or any combination thereof.

[0177] In some embodiments, the formulation may contain the anti-PSMA ADC of this disclosure at a concentration ranging from about 5 mg / mL to about 25 mg / mL. In some embodiments, the formulation may contain an anti-PSMA ADC at a concentration of about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, or about 25 mg / mL. In some embodiments, the formulation contains an anti-PSMA ADC at a concentration of about 9 mg / mL, about 10 mg / mL, or about 11 mg / mL. In some embodiments, the formulation contains an anti-PSMA ADC at a concentration of about 10 mg / mL. In some embodiments, the formulation contains an anti-PSMA ADC at a concentration of about 10 mg / mL ± 1 mg / mL.

[0178] In some embodiments, the anti-PSMA ADC formulation may contain a buffer. In some embodiments, the buffer is an acetate buffer, a succinate buffer, a histidine buffer, or a phosphate buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the formulation may have a histidine buffer concentration in the range of about 10 mM to about 50 mM. In some embodiments, the formulation may have a histidine buffer concentration in the range of about 10 mM to about 30 mM. In some embodiments, the formulation may have a histidine buffer concentration in the range of about 15 mM to about 25 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, or about 25 mM. In some embodiments, the formulation may have a histidine buffer concentration of about 20 mM. In some embodiments, the histidine is L-histidine. In some embodiments, the histidine buffer comprises L-histidine and a salt of L-histidine, such as L-histidine hydrochloride, and may be provided in water. Those skilled in the art can use various combinations of L-histidine and L-histidine hydrochloride concentrations to achieve the target pH of the histidine buffer.

[0179] In some embodiments, the anti-PSMA ADC formulation is characterized by having a pH value. In some embodiments, the formulation may have a pH in the range of about 5 to about 7.4. In some embodiments, the formulation may have a pH of up to about 7, up to about 6.5, up to about 6.2, or up to about 6. In some embodiments, the formulation may have a pH in the range of about 5.5 to about 6.5. In some embodiments, the formulation may have a pH in the range of about 5.4 to about 6.4. In some embodiments, the formulation may have a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.3, about 6.4, or about 6.5. In some embodiments, the formulation has a pH less than 6. In some embodiments, the formulation may have a pH in the range of about 5.5 to about 6.3. In some embodiments, the formulation may have a pH in the range of about 5.6 to about 6.2. In some embodiments, the formulation may have a pH of about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, or about 6.2. In some embodiments, the formulation may have a pH of about 5.9 ± 0.3.

[0180] In some embodiments, the anti-PSMA ADC formulation contains a water-soluble polymer. In some embodiments, the water-soluble polymer is polyvinylpyrrolidone, glycerol, trehalose, fructose, sucrose, glucose, or mannose; or a combination thereof. In some embodiments, the water-soluble polymer is a sugar. In some embodiments, the sugar is trehalose, fructose, sucrose, glucose, or mannose. In some embodiments, the formulation has a sugar concentration ranging from about 1% (w / v) to about 20% (w / v). In some embodiments, the formulation has a sugar concentration of up to about 15% (w / v). In some other embodiments, the formulation has a sugar concentration ranging from about 5% (w / v) to about 15% (w / v). In some embodiments, the formulation has a sugar concentration of up to about 10% (w / v). In some embodiments, the formulation has a sugar concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w / v). In some embodiments, the sugar is sucrose. In some embodiments, the formulation has a sucrose concentration ranging from about 1% (w / v) to about 20% (w / v). In some embodiments, the formulation has a sucrose concentration of up to about 15% (w / v). In some embodiments, the formulation has a sucrose concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w / v). In some other embodiments, the formulation has a sucrose concentration ranging from about 5% (w / v) to about 15% (w / v). In some embodiments, the formulation has a sucrose concentration of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w / v). In some embodiments, the formulation has a sucrose concentration of up to about 10% (w / v). In some embodiments, the formulation has a sucrose concentration in the range of about 5% (w / v) to about 10% (w / v). In some embodiments, the formulation has a sucrose concentration of about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% (w / v). In some embodiments, the formulation has a sucrose concentration of about 9% (w / v).

[0181] In some embodiments, the anti-PSMA ADC formulation may contain a surfactant. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 20. In some other embodiments, the surfactant is polysorbate 80. In some embodiments, the formulation has a surfactant concentration of up to about 1% (w / v). In some embodiments, the formulation has a surfactant concentration of up to about 0.1% (w / v). In some embodiments, the formulation has a surfactant concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the formulation has a surfactant concentration of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.10% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of up to about 1% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of up to about 0.1% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration in the range of about 0.01% (w / v) to about 0.1% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.10% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration in the range of about 0.001% (w / v) to about 0.02% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.001% (w / v), about 0.002% (w / v), about 0.003% (w / v), about 0.004% (w / v), about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.015% (w / v), or about 0.02% (w / v). In some embodiments, the formulation has a polysorbate 80 concentration of about 0.01% (w / v).

[0182] In some embodiments, an aqueous formulation is provided comprising the anti-PSMA ADC of this disclosure, histidine buffer, sucrose, and polysorbate 80. It should be understood that, as used herein, "aqueous formulation" refers to a formulation containing water. In some embodiments, the aqueous formulation comprises an anti-PSMA ADC at a concentration ranging from about 5 mg / mL to about 25 mg / mL; a histidine buffer at a concentration ranging from about 10 mM to about 50 mM; sucrose at a concentration ranging from about 1% (w / v) to about 20% (w / v); and polysorbate 80 at a concentration ranging from about 0.005% (w / v) to about 0.1% (w / v). In some embodiments, the aqueous formulation may have a pH ranging from about 5.6 to about 6.2. In some embodiments, the aqueous formulation may have a pH of about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, or about 6.2. In some embodiments, the anti-PSMA ADC is ARX517. In some embodiments, the histidine is L-histidine.

[0183] In some more specific embodiments, an aqueous formulation comprising an anti-PSMA ADC at a concentration of about 10 mg / mL; a histidine buffer at a concentration of about 20 mM; sucrose at a concentration of about 9% (w / v); and polysorbate 80 at a concentration of about 0.01% (w / v); wherein the anti-PSMA ADC is ARX517, and the aqueous formulation has a pH of about 5.9 ± 0.3. In some embodiments, the histidine is L-histidine.

[0184] In some more specific embodiments, an aqueous formulation is provided, which is substantially composed of or consists of: an anti-PSMA ADC at a concentration of about 10 mg / mL; histidine at a concentration of about 20 mM; sucrose at a concentration of about 9% (w / v); and polysorbate 80 at a concentration of about 0.01% (w / v); and water; wherein the anti-PSMA ADC is ARX517, and the aqueous formulation has a pH of about 5.9 ± 0.3. In some embodiments, the histidine is L-histidine.

[0185] In some embodiments, pharmaceutical compositions containing the anti-PSMA ADC of this disclosure (such as liquid formulations) are preservative-free.

[0186] In some embodiments, the formulation is a liquid formulation. In some embodiments, the liquid formulation can be stored at room temperature. In some other embodiments, the liquid formulation can be stored frozen. In some embodiments, the liquid formulation can be stored frozen at -20°C. In still other embodiments, the liquid formulation can be stored refrigerated. In some embodiments, the liquid formulation can be refrigerated at 5°C ± 3°C. The formulation can be contained in a glass vial. For example, a glass vial can contain 5.0 mL of ARX517 at 10 mg / mL in a frozen solution. In another example, a glass vial can contain 5.0 mL of ARX517 at 10 mg / mL in a refrigerated solution.

[0187] The composition may be stored in vials or cartridges, pen delivery devices, syringes, intravenous administration tubing, or intravenous administration bags, but is not limited thereto. The composition may be diluted with a diluent such as water, pH-adjusted water, or water for injection. The composition may be diluted into an IV bag, for example, in 0.9% saline for injection. For example, a liquid formulation containing about 10 mg / mL of an anti-PSMA ADC; about 20 mM of histidine; about 9% (w / v) of sucrose; and about 0.01% (w / v) of polysorbate 80; and water; wherein the anti-PSMA ADC is ARX517, and the aqueous formulation has a pH of about 5.9 ± 0.3, may be added to an infusion bag containing 0.9% saline for injection.

[0188] As will be understood by those skilled in the art, biotherapeutic proteins may contain charged variants. Therefore, compositions comprising an ADC of this disclosure (such as ARX517) may contain one or more charged variants, as further discussed herein. In some embodiments, the composition is a liquid formulation of this disclosure. In some embodiments, the composition is an ARX517 solution for intravenous infusion.

[0189] Analysis of charged variants is a regulatory requirement for biotherapeutic proteins. Methods for analyzing charged variants of biotherapeutic proteins (such as mAbs or ADCs) are known in the art and include ion-exchange chromatography, such as cation-exchange chromatography (CEX) or anion-exchange chromatography, which is typically performed using a salt gradient. CEX is a type of ion-exchange chromatography that separates molecules based on their net surface charge, such as proteins.

[0190] As will be further understood by those skilled in the art, the isoelectric point (pI) of a protein is the pH value at which the protein carries no net charge. The net surface charge of a protein varies with pH as follows: i) the protein will have no net charge when the pH (e.g., the pH of a solution containing the protein) is the same as the protein's pI; ii) the protein will have a net positive charge when the pH (e.g., the pH of a solution containing the protein) is less than the protein's pI; and iii) the protein will carry a net negative charge when the pH (e.g., the pH of a solution containing the protein) is greater than the protein's pI. By using a negatively charged ion-exchange stationary phase, such as a resin with an affinity for molecules with a net positive surface charge, CEX can be used to separate mixtures of proteins with different pI values.

[0191] Because the pI of a protein of interest can be predicted based on its primary amino acid sequence or determined by methods known to those skilled in the art (such as isoelectric focusing), a suitable buffer and buffer pH can be chosen to ensure the known net charge of the protein of interest. When the protein of interest carries a net positive charge at the working buffer pH, a negatively charged cation exchange resin can be chosen such that the negatively charged resin will attract the positive surface charge of the protein in the test sample. Proteins with different pI values ​​in the sample will have different degrees of charge at a given pH and therefore will have different affinities for the positively charged surface groups on the particles of the anion exchange medium. Thus, different proteins will bind to the resin with different strengths, thereby promoting their separation. In a non-limiting example, if a cation exchange resin is used at pH 7.5, generally all proteins with pI > 7.5 will carry a net positive charge and will bind to the negatively charged resin. A salt gradient can then be used to separate the protein of interest from other bound proteins. The proteins will be eluted in a sequence that depends on their net surface charge. Proteins with a pI value close to 7.5 will elute at lower ionic strengths (e.g., lower salt concentrations), while proteins with very high pI values ​​will elute at higher ionic strengths (e.g., higher salt concentrations).

[0192] Compositions containing monoclonal antibodies (mAbs) or ADCs may contain variants of the mAb or its ADC resulting from post-translational modifications and / or degradation events. Typical post-translational modifications may include N-terminal glutamine (Gln) cyclization to pyroglutamic acid (pyroGlu); removal of the C-terminal lysine residue (Lys) from the heavy chain; and / or glycosylation of conserved asparagine (Asn) in the CH2 domain with a neutral oligosaccharide. Such variants can be observed when analyzing compositions containing mAbs or ADCs using CEX chromatography. These variants are generally referred to as acidic or basic substances compared to the main substance. When using CEX chromatography, acidic substances are variants that elute earlier than the main substance, and basic substances are variants that elute later than the main substance. The corresponding peaks in the CEX chromatogram can be referred to as acidic peaks, corresponding to acidic substances; main peaks, corresponding to the main substance; and basic peaks, corresponding to the elution of basic substances. (See, for example, Du, Y. et al., Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs, Sep-October 2012; 4(5):578-85. doi: 10.4161 / mabs.21328.Epub July 23, 2012, PMID: 22820257; PMCID:PMC3499298).

[0193] In some aspects, this disclosure provides compositions comprising anti-PSMA ADCs, wherein the composition comprises a major substance of an anti-PSMA ADC. In some embodiments, the major substance of the anti-PSMA ADC has a pI of about 8.3. For example, ARX517 has a pI of 8.3 as determined by imaging capillary isoelectric focusing (iCIEF; see, e.g., Example 18). In some embodiments, the composition comprises an anti-PSMA ADC major substance and one or more variants thereof. In some embodiments, one or more variants comprise an acidic substance of an anti-PSMA ADC, a basic substance of an anti-PSMA ADC, or a combination thereof. In some embodiments, the acidic substance of the anti-PSMA ADC has a pI of about 8.1, and the basic substance of the anti-PSMA ADC has a pI of about 8.4. For example, the acidic and basic substances of ARX517 have pIs of 8.1 and 8.4, respectively, as determined by iCIEF. In some embodiments, the anti-PSMA ADC is ARX517. In some embodiments, the composition is an ARX517 solution for intravenous infusion as disclosed herein.

[0194] In some embodiments, the anti-PSMA ADC of this disclosure or a composition comprising the anti-PSMA ADC of this disclosure is characterized by an ADC charge change curve. In some embodiments, the composition is characterized by having an anti-PSMA ADC main substance and one or more variants thereof, or the composition comprises an anti-PSMA ADC main substance and one or more variants thereof, wherein the one or more variants comprise an anti-PSMA ADC acidic substance and an anti-PSMA ADC basic substance. In some embodiments, respectively, the anti-PSMA ADC main substance has a pI of about 8.3, the anti-PSMA ADC acidic substance has a pI of about 8.1, and the anti-PSMA ADC basic substance has a pI of about 8.4. In some embodiments, the anti-PSMA ADC is ARX517. In some embodiments, the composition is an ARX517 solution for intravenous infusion as disclosed herein.

[0195] In some embodiments, the composition is characterized by having an anti-PSMA ADC main substance, or the composition comprises an anti-PSMA ADC main substance, wherein the anti-PSMA ADC main substance is present in an amount of at least about 35%. In some other embodiments, the composition is characterized by having an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, or the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of at least about 35%, the anti-PSMA ADC acidic substance is present in an amount of up to about 45%, and the anti-PSMA ADC basic substance is present in an amount of up to about 35%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 35% to about 70%, the anti-PSMA ADC acidic substance is present in an amount of about 20% to about 45%, and the anti-PSMA ADC basic substance is present in an amount of about 5% to about 35%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 35% to about 60%, the anti-PSMA ADC acidic substance is present in an amount of about 20% to about 45%, and the anti-PSMA ADC basic substance is present in an amount of about 5% to about 30%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 35% to about 55%, the anti-PSMA ADC acidic substance is present in an amount of about 25% to about 45%, and the anti-PSMA ADC basic substance is present in an amount of about 5% to about 25%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some other embodiments, the composition is characterized by having an anti-PSMA ADC main substance, or the composition comprises an anti-PSMA ADC main substance, wherein the anti-PSMA ADC main substance is present in an amount of at least about 40%.In some embodiments, the composition is characterized by having an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, or the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of at least about 40%, the anti-PSMA ADC acidic substance is present in an amount of at most about 40%, and the anti-PSMA ADC basic substance is present in an amount of at most about 30%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some other embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 40% to about 70%, the anti-PSMA ADC acidic substance is present in an amount of about 20% to about 40%, and the anti-PSMA ADC basic substance is present in an amount of about 5% to about 30%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In still other embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 40% to about 60%, the anti-PSMA ADC acidic substance is present in an amount of about 25% to about 40%, and the anti-PSMA ADC basic substance is present in an amount of about 5% to about 20%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some more specific embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 45% to about 60%, the anti-PSMA ADC acidic substance is present in an amount of about 25% to about 35%, and the anti-PSMA ADC basic substance is present in an amount of about 10% to about 20%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%. In some other embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of at least about 50%, the anti-PSMA ADC acidic substance is present in an amount of at most about 35%, and the anti-PSMA ADC basic substance is present in an amount of at most about 20%; wherein the sum of the percentages of the main substance, the acidic substance, and the basic substance is 100%.In some other embodiments, the composition comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance, wherein the anti-PSMA ADC main substance is present in an amount of about 50% to about 55%, the anti-PSMA ADC acidic substance is present in an amount of about 30% to about 35%, and the anti-PSMA ADC basic substance is present in an amount of about 12% to about 17%; wherein the sum of the percentages of the main substance, acidic substance, and basic substance is 100%. In some embodiments, the percentages of the main substance, acidic substance, and basic substance are based on the UV area percentage at 214 nm of the integral of the corresponding main substance elution peak, acidic substance elution peak, and basic substance elution peak in the cation exchange chromatogram. In some embodiments, the acidic substance elution peak includes or consists of all peaks in the cation exchange chromatogram eluted before the main peak, and the basic substance elution peak includes or consists of all peaks in the cation exchange chromatogram eluted after the main peak. In some embodiments, the cation exchange chromatogram is obtained from the CEX method disclosed in Example 17. In some embodiments, the charge change profile of the anti-PSMA ADC or a composition containing an anti-PSMA ADC is determined by the CEX method as disclosed in Example 17. In some embodiments, the anti-PSMA ADC is ARX517. In some embodiments, the composition is an ARX517 solution for intravenous infusion as disclosed in Example 14.

[0196] The pharmaceutical compositions of the present invention can be administered as a single dose, or one or more subsequent doses can be administered minutes, days, or weeks after the first administration. Further administration may be considered as needed to treat, alleviate, or prevent cancer, symptoms, conditions, or diseases, including prostate cancer.

[0197] Therapeutic uses of anti-PSMA antibodies and ADCs This disclosure provides anti-PSMA ADCs for inhibiting, preventing, or treating PSMA-related diseases or cancers, and relates to methods of treating subjects who have or are at risk of developing PSMA-related diseases or cancers. These uses and methods may include dosing regimens for inhibiting, preventing, or treating PSMA-related diseases or cancers. In some embodiments, the use or method of treatment includes administering the anti-PSMA ADC of this disclosure to a subject in need. The use or method of treatment may also include treating the subject with additional therapies, as further disclosed herein.

[0198] The anti-PSMA antibody or anti-PSMA ADC of this disclosure can be used to treat a variety of diseases, conditions, ailments, or cancers. This disclosure includes compositions and methods for treating mammals (e.g., humans) that are at risk of, have, or have already had a disease or ailment (such as cancer) that responds to PSMA overexpression, amplification, mutation, and / or targeted therapy. In some embodiments, compositions comprising an effective amount of the ADC of this disclosure can be used to reduce or inhibit tumor growth or progression in cancers or cancer cells expressing antigens (such as cancers or cancer cells expressing prostate-specific antigens).

[0199] The compositions disclosed herein can be used to modulate immune responses. Modulation of immune responses may include stimulating, activating, increasing, enhancing, or upregulating immune responses. Modulation of immune responses may also include suppressing, inhibiting, preventing, reducing, or downregulating immune responses.

[0200] Administration of ADCs can result in short-term effects, i.e., an immediate beneficial effect on several observed clinical parameters, and this may occur 12 or 24 hours after administration, and / or can result in long-term effects, such as a beneficial slowing of tumor growth progression or a reduction in tumor size. The ADCs of this disclosure can be administered in any manner known to those skilled in the art, and can advantageously be administered via infusion, such as by arterial, intraperitoneal, or intravenous injection and / or infusion at a dose sufficient to achieve the desired pharmacological effect. In some embodiments, the ADC or a composition or formulation containing the ADC is administered orally, intradermally, intratumorally, intravenously, or subcutaneously. In some embodiments, the ADC or a composition or formulation containing the ADC is administered intravenously.

[0201] This disclosure provides a method of treating a disease or condition, such as a tumor or cancer, by administering a therapeutically effective amount of the disclosed anti-PSMA ADC or a pharmaceutical composition comprising the disclosed anti-PSMA ADC to a patient in need (e.g., a human subject).

[0202] In some embodiments, the anti-PSMA ADC of this disclosure can be used to inhibit the growth of cancer cells. In some embodiments, the cancer cells are prostate cancer cells, bladder cancer cells, pancreatic cancer cells, liver cancer cells, lung cancer cells, kidney cancer cells, sarcoma cells, breast cancer cells, brain cancer cells, neuroendocrine cancer cells, colon cancer cells, testicular cancer cells, or melanoma cells. In some more specific embodiments, the cancer cells are prostate cancer cells.

[0203] In some embodiments, the anti-PSMA ADC of this disclosure can be used to treat a subject's cancer, wherein the cancer is prostate cancer. In some embodiments, the prostate cancer is metastatic castration-refractory prostate cancer.

[0204] In some other embodiments, the anti-PSMA ADC of this disclosure can be used to treat a subject's cancer, wherein the cancer is non-prostate cancer. In some embodiments, non-prostate cancer is bladder cancer, pancreatic cancer, liver cancer, lung cancer, kidney cancer, sarcoma, breast cancer, brain cancer, neuroendocrine cancer, colon cancer, testicular cancer, or melanoma.

[0205] The dose of the anti-PSMA ADC of this disclosure administered to a subject is sufficient to elicit a favorable response in the subject over time. The anti-PSMA ADC dose may be administered as a bolus and / or as an infusion for a duration clinically necessary, e.g., a period ranging from minutes to hours, for example, up to 24 hours. In some embodiments, the anti-PSMA ADC is administered via intravenous infusion and lasts for about 30 minutes to about 120 minutes. In some embodiments, the anti-PSMA ADC is administered via intravenous infusion and lasts for about 90 minutes. In some embodiments, the anti-PSMA ADC is administered via intravenous infusion and lasts for about 60 minutes. If necessary, the anti-PSMA ADC administration may be repeated once or several times. In some embodiments, the infusion time of the anti-PSMA ADC is reduced within the range of subsequent administrations. In some embodiments, a first anti-PSMA ADC dose is administered to the subject via intravenous infusion for about 90 minutes, and a second anti-PSMA ADC dose is subsequently administered via intravenous infusion for about 60 minutes. The anti-PSMA ADC dose may be an effective amount or a dosage. In some embodiments, the dose is administered more than once at one-hour intervals. In some embodiments, the dose is administered more than once at daily intervals. In another embodiment, the dose is administered more than once at weekly intervals. In another embodiment, the dose is administered at weekly intervals. In another embodiment, the dose is administered at weekly intervals. In another embodiment, the dose is administered at weekly intervals. In another embodiment, the dose is administered at weekly intervals. In another embodiment, the dose is administered at weekly intervals. In another embodiment, the dose is administered at weekly intervals.

[0206] In some embodiments, an effective amount of anti-PSMA ADC is sufficient to: 1) delay or inhibit cancer progression in the subject; 2) increase the subject's survival compared to median survival in subjects with metastatic castration-refractory prostate cancer that has progressed after prior taxane therapy and who have not been treated with anti-PSMA ADC; 3) reduce the circulating tumor cell (CTC) level in the subject compared to baseline; and 4) reduce or stabilize serum prostate-specific antigen (PSA) levels in the subject compared to baseline PSA levels. In another embodiment, treatment with the anti-PSMA ADC of this disclosure results in an improvement in the subject's quality of life compared to the subject's quality of life before treatment with the anti-PSMA ADC. When assessing any or more of the foregoing changes, in some embodiments, the first time point may be at baseline or before administration of any PSMA ADC, while the second time point is any time after administration of the anti-PSMA ADC. In other embodiments, the first time point occurs after administration of the anti-PSMA ADC, and the second time point occurs any time after the first time point. In some implementations, the first time point occurs before a specific dose of the anti-PSMA ADC, and the second time point occurs after that specific dose of the anti-PSMA ADC.

[0207] In some embodiments, treatment of a subject with the anti-PSMA ADC of this disclosure results in a delay or inhibition of cancer progression in the subject. As used herein, “delay or inhibition of cancer progression” is intended to refer to any slowing or cessation of cancer progression in the subject. A slowing or cessation of cancer progression includes a reduction or stabilization of the number of cancer cells, the number of tumors, and / or the number of metastases in the subject. A slowing or cessation is also intended to include a reduction or stabilization of the size (e.g., length or volume) of the tumor and / or the size of the metastases in the subject. In other embodiments, the delay or inhibition of cancer progression is demonstrated by changes in radiographic images of the tumor burden compared to baseline radiographic images of the subject prior to anti-PSMA ADC treatment. Methods for assessing changes in radiographic images include, for example, computed axial computed tomography (CT) scans and magnetic resonance imaging (MRI). Methods for assessing cancer progression in subjects will be readily apparent to those skilled in the art.

[0208] In another embodiment, treatment with an anti-PSMA ADC resulted in an increased survival in the subject, specifically an increased survival compared to the median survival of subjects with taxane-resistant cancer expressing anti-PSMA who were not treated with an anti-PSMA ADC. In some embodiments, the subject's survival increased by four weeks. In another embodiment, the subject's survival increased by six weeks. In another embodiment, the subject's survival increased by two months. In another embodiment, the subject's survival increased by four months. In another embodiment, the subject's survival increased by six months. In another embodiment, the subject's survival increased by eight months. In another embodiment, the subject's survival increased by ten months. In another embodiment, the subject's survival increased by twelve months. In another embodiment, the subject's survival increased by fourteen months. In some embodiments, compared to those not treated with anti-PSMA ADCs... Compared to median survival, subjects treated with ADC who had metastatic castration-refractory prostate cancer that had progressed after prior taxane therapy experienced survival increases of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 56, 58, or 60 weeks or longer. In some other implementations, survival is extended by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 56, 58, or 60 weeks or longer compared to the expected survival of subjects before treatment with anti-PSMAADC. In other implementations, depending on any of the foregoing comparisons, the survival of the subjects is increased by 6 months, 10 months, 12 months, 14 months, 16 months, 18 months, 20 months, 22 months, 24 months, 26 months, 28 months, 30 months, 32 months, 34 months, 36 months, 38 months, 40 months, 42 months, 44 months, 46 months, 48 ​​months, 50 months, 52 months, 54 months, 56 months, 58 months, or 60 months or longer.

[0209] In some embodiments, subjects treated with the anti-PSMA ADC have metastatic castration-refractory prostate cancer that has progressed after prior taxane therapy, and the increase in survival is compared to the median survival (v) of subjects with metastatic castration-refractory prostate cancer that has progressed after prior taxane therapy and has not been treated with the ADC. In another embodiment, subjects treated with the anti-PSMA ADC have metastatic castration-refractory prostate cancer that has progressed after prior taxane therapy, and the increase in survival is compared to the expected survival of the subject before treatment with the ADC.

[0210] In some embodiments, treatment with an anti-PSMA ADC results in a decrease in the circulating levels of circulating tumor cells (CTCs) compared to baseline levels. In other embodiments, ADC treatment results in a decrease or stabilization (no significant increase or decrease) in serum PSA levels compared to baseline PSA levels. Methods for assessing circulating CTC levels or serum PSA levels are well known to those skilled in the art.

[0211] In some embodiments, treatment of subjects with the anti-PSMA ADC of this disclosure results in a reduction in tumor volume. In some embodiments, the tumor volume reduction is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, and 48%. The percentages are 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%. In some implementations, the tumor volume is reduced by at least approximately 99%.

[0212] In another embodiment, administration of the anti-PSMA ADC to the subject results in a weight gain in the subject (compared to the subject's weight before administration of the PSMA ADC). The weight gain may be caused by a single administration of the PSMA ADC or by a course of treatment with the anti-PSMA ADC (i.e., more than one administration). In some embodiments, the weight gain is about 5% to about 10%. In another embodiment, the weight gain is about 10% to about 20%. In yet another embodiment, the weight gain is about 15%. In another embodiment, the weight gain is about 25%. In yet another embodiment, the weight gain is about 30%, about 35%, or more.

[0213] In some implementations, the dose of the anti-PSMA ADC administered to the subject can be selected based on various parameters, specifically the administration method used and the subject's condition. Other factors include the desired duration of treatment. If the response in the subject is insufficient at the initial dose, a higher dose (or an even higher dose effectively delivered via a different, more localized route) can be used up to the extent tolerated by the patient.

[0214] In some implementations, the anti-PSMA ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each of the heavy chains at position 114 according to the Kabat number, and wherein a drug linker is conjugated to each of the pAFs via an oxime bond, wherein each of the drug linkers is amberstatin269 (AS269) having the following structure: .

[0215] In some embodiments, each heavy chain includes the heavy chain variable region of SEQ ID NO: 1. In some embodiments, each light chain includes the light chain variable region of SEQ ID NO: 2. In some embodiments, each heavy chain includes the heavy chain variable region of SEQ ID NO: 1, and each light chain includes the light chain variable region of SEQ ID NO: 2.

[0216] In some implementations, the anti-PSMA ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain contains the heavy chain variable region of SEQ ID NO:1, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, wherein each light chain contains the light chain variable region of SEQ ID NO:2; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0217] In some embodiments, each heavy chain has the amino acid sequence of SEQ ID NO: 8. In some embodiments, each light chain has the amino acid sequence of SEQ ID NO: 9. In some embodiments, each heavy chain has the amino acid sequence of SEQ ID NO: 8, which contains pAF at Kabat amino acid position 114 (i.e., amino acid position 116 of SEQ ID NO: 8), and each light chain has the amino acid sequence of SEQ ID NO: 9.

[0218] In some implementations, the PSMA-resistant ADC is an ARX517. In some implementations, the PSMA-resistant ADC includes: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO:8, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, and each light chain has the amino acid sequence of SEQ ID NO:9; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: .

[0219] In some embodiments, the effective dose of the anti-PSMA ADC administered to a human subject is a dose ranging from about 0.32 mg / kg to about 10 mg / kg of human subject body weight. In other embodiments, the effective dose is a dose ranging from about 0.32 mg / kg to about 5 mg / kg, about 1.4 mg / kg to about 5 mg / kg, about 1.4 mg / kg to about 3.4 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 5 mg / kg, or about 2 mg / kg to about 3.4 mg / kg of human subject body weight. In other embodiments, the dose is about 0.32 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 7 mg / kg, or about 10 mg / kg of human subject body weight. In yet another embodiment, the dose is about 0.32 mg / kg or about 0.64 mg / kg. The dosage is approximately 1.07 mg / kg, approximately 1.4 mg / kg, approximately 1.7 mg / kg, approximately 2.0 mg / kg, approximately 2.4 mg / kg, approximately 2.88 mg / kg, approximately 3.4 mg / kg, or approximately 4.5 mg / kg of human subject weight. In some embodiments, the dosage is greater than 2 mg / kg of human subject weight. In some embodiments, the dosage is approximately 2.1 mg / kg, approximately 2.2 mg / kg, approximately 2.3 mg / kg, approximately 2.4 mg / kg, approximately 2.5 mg / kg, approximately 2.6 mg / kg, approximately 2.7 mg / kg, approximately 2.8 mg / kg, approximately 2.9 mg / kg, or approximately 3.0 mg / kg of human subject weight. In some embodiments, the dosage is greater than 3 mg / kg of human subject weight. In some embodiments, the dosage is approximately 3.1 mg / kg, approximately 3.2 mg / kg, approximately 3.3 mg / kg, or approximately 3.4 mg / kg of human subject weight. In some embodiments, the dosage is about 3.5 mg / kg, about 3.6 mg / kg, about 3.7 mg / kg, about 3.8 mg / kg, about 3.9 mg / kg, about 4.0 mg / kg, about 4.1 mg / kg, about 4.2 mg / kg, about 4.3 mg / kg, about 4.4 mg / kg, or about 4.5 mg / kg of human subject weight. In some embodiments, the dosage is administered to human subjects on a dosing schedule of about once a week. In some embodiments, the dosage is administered to human subjects on a dosing schedule of about once every two weeks. In some embodiments, the dosage is administered to human subjects on a dosing schedule of about once every three weeks. In some embodiments, the dosage is administered to human subjects on a dosing schedule of about once every four weeks. In some embodiments, the anti-PSMA ADC is ARX517.

[0220] In some embodiments, the effective amount of the anti-PSMA ADC of this disclosure administered to a human subject is a dose ranging from about 0.05 mg / kg to about 10 mg / kg of human subject weight, or any value therebetween. In some other embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 2 mg / kg to about 10 mg / kg of human subject weight, or any value therebetween. In some embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 1.4 mg / kg to about 5 mg / kg of human subject weight, or any value therebetween. In some embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 1.4 mg / kg to about 5 mg / kg of human subject weight, or any value therebetween. In some embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 1.4 mg / kg to about 3.4 mg / kg of human subject weight, or any value therebetween. In some embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 2.0 mg / kg to about 5 mg / kg of the human subject's body weight, or any value therebetween. In some embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 2.1 mg / kg to about 4.5 mg / kg of the human subject's body weight, or any value therebetween. In some embodiments, the effective amount of the anti-PSMA ADC administered to a human subject is a dose ranging from about 3.0 mg / kg to about 4.5 mg / kg of the human subject's body weight, or any value therebetween.In some implementations, the effective amount of the anti-PSMA ADC is approximately 1.0 mg / kg, approximately 1.2 mg / kg, approximately 1.3 mg / kg, approximately 1.4 mg / kg, approximately 1.5 mg / kg, approximately 1.6 mg / kg, approximately 1.7 mg / kg, approximately 1.8 mg / kg, approximately 1.9 mg / kg, approximately 2.0 mg / kg, approximately 2.1 mg / kg, approximately 2.2 mg / kg, approximately 2.3 mg / kg, approximately 2.4 mg / kg, approximately 2.5 mg / kg, approximately 2.6 mg / kg, approximately 2.7 mg / kg, approximately 2.8 mg / kg, approximately 2.9 mg / kg, and approximately 3.0 mg / kg. Doses are administered at approximately 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, or 5 mg / kg of human subject weight. In some embodiments, the dose is administered to human subjects on a dosing schedule of approximately once a week. In some embodiments, the dose is administered to human subjects on a dosing schedule of approximately once every two weeks. In some embodiments, the dose is administered to human subjects on a dosing schedule of approximately once every three weeks. In some embodiments, the dose is administered to human subjects on a dosing schedule of approximately once every four weeks. In some implementations, the anti-PSMA ADC is ARX517.

[0221] In some embodiments, the effective amount of the anti-PSMA ADC of this disclosure administered to a human subject is a dose greater than 2.0 mg / kg and up to about 10.0 mg / kg of human subject body weight. In some embodiments, the effective amount is a dose of at least about 2.4 mg / kg of human subject body weight. In some embodiments, the effective amount is a dose of at least about 2.4 mg / kg and up to about 5.0 mg / kg of human subject body weight. In some embodiments, the effective amount is a dose of at least about 2.4 mg / kg and up to about 4.0 mg / kg of human subject body weight. In some embodiments, the effective amount is a dose of about 2.4 mg / kg, about 2.9 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, or about 3.9 mg / kg of human subject body weight. In some embodiments, the effective amount is a dose of at least about 2.4 mg / kg and up to about 3.5 mg / kg of human subject body weight. In some embodiments, the effective dose is a dose of at least about 2.4 mg / kg and at most about 3.4 mg / kg of human subject weight. In some embodiments, the effective dose is a dose of at least about 2.4 mg / kg and at most about 3.2 mg / kg of human subject weight. In some embodiments, the effective dose is a dose of at least about 2.4 mg / kg and at most about 3.0 mg / kg of human subject weight. In some embodiments, the effective dose is a dose of at least about 2.8 mg / kg and at most about 3.4 mg / kg of human subject weight. In some embodiments, the effective dose is a dose of at least about 2.8 mg / kg and at most about 3.2 mg / kg of human subject weight. In some embodiments, the effective dose is administered to human subjects on a dosing schedule of approximately once a week. In some embodiments, the effective dose is administered to human subjects on a dosing schedule of approximately once every two weeks. In some embodiments, the effective dose is administered to human subjects on a dosing schedule of approximately once every three weeks. In some embodiments, the effective dose is administered to human subjects on a dosing schedule of approximately once every four weeks.

[0222] In some embodiments, the effective amount of the anti-PSMA ADC of this disclosure administered to a human subject is a dose in the range of about 2.0 mg / kg to about 4.0 mg / kg of human subject body weight, wherein the dose is administered to the human subject once every three weeks, and wherein the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.0 mg / kg of human subject body weight, wherein the dose is administered to the human subject once every three weeks, and wherein the anti-PSMA ADC is ARX517. In some embodiments, the dose is greater than 2.0 mg / kg of human subject body weight, wherein the dose is administered to the human subject once every three weeks, and wherein the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.1 mg / kg of human subject body weight, wherein the dose is administered to the human subject once every three weeks, and wherein the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.2 mg / kg of human subject body weight, wherein the dose is administered to the human subject once every three weeks, and wherein the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.3 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.4 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.5 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is greater than 2.5 mg / kg of human subject body weight. In some embodiments, the dose is about 2.6 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 2.7 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 2.8 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 2.9 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 3.0 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517.In some embodiments, the dose is about 3.1 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.2 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.3 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.4 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.5 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.6 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.7 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.8 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 3.9 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is about 4.0 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 4.1 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 4.2 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 4.3 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517. In some embodiments, the dose is approximately 4.4 mg / kg of human subject body weight, administered to human subjects every three weeks, and the anti-PSMA ADC is ARX517.In some implementations, the dose is approximately 4.5 mg / kg of human subject body weight, wherein the dose is administered to the human subject once every three weeks, and wherein the anti-PSMA ADC is ARX517.

[0223] In other embodiments, based on the composition, the dosage may be delivered continuously, such as by a continuous pump, or at periodic intervals. Those skilled in the art can determine the desired time intervals for multiple doses of a particular composition without excessive experimentation. Other regimens for administering the provided composition will be known to those skilled in the art, wherein the dosage, administration schedule, application site, manner of administration, etc., differ from those described above. In some embodiments, the dosage regimen is a single intravenous dose. In some other embodiments, the dosage regimen begins with a single intravenous dose, followed by a series of additional single intravenous doses, each administered approximately every 3 weeks.

[0224] In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment provides a serum terminal half-life of anti-PSMA ADC for at least about 5 days. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment provides a serum terminal half-life of anti-PSMA ADC in the range of about 5 days to about 10 days. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment provides a serum terminal half-life of anti-PSMA ADC for at least about 6 days. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment provides a serum terminal half-life of anti-PSMA ADC for at least about one week. In some embodiments, following administration of an effective amount of anti-PSMA ADC to a human subject, the treatment provides a serum terminal half-life of anti-PSMA ADC in the range of about 6 days to about 10 days.

[0225] In some implementations, following administration of an effective amount of the anti-PSMA ADC to a human subject, the treatment provides at least approximately 5 days for the free payload to reach its maximum serum concentration (Tmax), wherein the free payload has the following structure: ; Or its salts. In some embodiments, the treatment provides at least about 6 days of free payload Tmax after administration of an effective amount of anti-PSMA ADC to a human subject. In some embodiments, the treatment provides about one week of free payload Tmax after administration of an effective amount of anti-PSMA ADC to a human subject.

[0226] In some embodiments, after administration of an effective amount of anti-PSMA ADC to a human subject, the treatment method provides a maximum free payload serum concentration (Cmax) of up to about 1 ng / mL. In some embodiments, after administration of an effective amount of anti-PSMA ADC to a human subject, the treatment method provides a free payload Cmax of up to about 0.5 ng / mL, up to about 0.4 ng / mL, up to about 0.3 ng / mL, or up to about 0.2 ng / mL. In some embodiments, after administration of an effective amount of anti-PSMA ADC to a human subject, the treatment method provides a free payload Cmax in the range of about 0.01 ng / mL to about 0.3 ng / mL.

[0227] In some implementations, after administration of an effective amount of anti-PSMA ADC to a human subject, the treatment method provides a maximum serum concentration (Cmax) of at least about 20 μg / ml (20 μg / mL), at least about 25 μg / ml (20 μg / mL), at least about 30 μg / ml, at least 35 μg / ml, at least about 40 μg / ml, at least about 45 μg / ml, at least about 50 μg / ml, at least about 55 μg / ml, or at least about 60 μg / ml of anti-PSMA ADC. (See, for example, Figure 16B ).

[0228] In some cases, the PSMA antibody, variant, or anti-PSMA ADC composition disclosed herein may be used in combination with additional therapies or treatments, including but not limited to surgery, radiation, cryosurgery, hyperthermia, hormone therapy, chemotherapy, vaccines, and other immunotherapies. In some embodiments, such additional treatment may include therapeutic agents such as chemotherapeutic agents, hormones, antitumor agents, immunostimulants, immunomodulators, corticosteroids, or combinations thereof. In some embodiments, the hormone agent is enzalutamide.

[0229] In other embodiments, the anti-PSMA ADC of this disclosure may be administered in conjunction with one or more immunostimulants to induce or enhance an immune response. Immunostimulants that stimulate specific branches of the immune system, such as natural killer (NK) cells mediating antibody-dependent cytotoxicity (ADCC), include, but are not limited to, IL-2, immunostimulatory oligonucleotides (e.g., CpG motifs), α-interferon, γ-interferon, and tumor necrosis factor α (TNFα). In other embodiments, the anti-PSMA ADC of this disclosure may be administered in conjunction with one or more immunomodulators, including but not limited to cytokines, chemokines (including but not limited to SLC5 ELC, MIP3α, MIP3β, IP-IO, MIG, and combinations thereof). Other therapeutic agents may be vaccines that immunize a subject against PSMA. In some embodiments, such vaccines include an antigen (such as a PSMA dimer) and optionally one or more adjuvants to induce or enhance an immune response. Many types of adjuvants are well known in the art.

[0230] Chemotherapy agents or any agents involved in treating, alleviating, or preventing a disease, symptom, or cancer in a subject of need may also be administered in combination with the anti-PSMA ADC of this disclosure. Chemotherapy agents may include, but are not limited to, erlotinib (TARCEVA). ® Genentech / OSI Pharm.), bortezomib (VELCADE) ® Millennium Pharm.), fulvestrant (FASLODEX) ® AstraZeneca), sunitin (SU11248, Pfizer), letrozole (FEMARA) ® Novartis), imatinib mesylate (GLEEVEC) ® Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin) ® Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus), RAPAMUNE ® Wyeth), lapatinib (TYKERB) ®GSK572016, GlaxoSmithKline, lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA) ® AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN ® Cyclophosphamides; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; antifolate and antitumor agents, such as pemetrexed (ALIMTA). ®Eli Lilly), aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trihydroxymethylmelamine; anechoic acid lactones (especially bulbatacin and bulbatacinone); camptothecin (including the synthetic analogue topotecan); lichenin; callystatin; CC -1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); sea hare toxin; bicarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard. Mustards, such as chlornaphazine, cholophosphamide, estradiol, ifosfamide, dichloroethyl methylamine, mechlorethamine oxide hydrochloride, melphalan, novobichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorhexidine, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics, kazimidic acid, kazimidic acid γ1I, and kazimidic acid ωI1; danendomycin, including danendomycin A; bisphosphonates, such as clophosphonate; and esperamicin.And new carcinogen chromophores and related pigment proteins enediyne antibiotic chromophores, aclacinomysins, actinomycin, antramycin, diazoserine, bleomycins, cactinomycin C, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN; ®Doxorubicin (including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrole doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zineb ostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluorouridine; androgen derivatives, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; antiandrogens (e.g., enzalutamide) or androgen deprivation therapy; antiadrenergics such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; acridine; bestrabucil;Bisantrene; Edatrix; Defofamide; Demecolcine; Deacon; Elformithine; Elliptinium acetate; Epothilone; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Loninidainine; Maytansinoids, such as Maytansin and Ansamitocins; Mitoguazone; Mitoantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Loxoantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK; ® Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; lizofuran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactone; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL). ® , Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ™ Nanoparticle formulations free of Cremophor, albumin, palitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and TAXOTERE ®Docetaxel (Rhone-Poulenc Rorer, Antony, France); Chloranbucil; GEMZAR ® Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine ® Vinorelbine; novantrone; teniposide; idatraxa; donomycin; aminopterin; capecitabine; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances.

[0231] The anti-PSMA ADC of the present invention can be used in combination with any agent involved in the treatment, mitigation, improvement, or prevention of prostate cancer in subjects of need, including but not limited to hormone therapy or chemotherapy. For example, the hormone therapy enzalutamide has been shown in vitro to increase cell surface PSMA expression by approximately 3-fold (Murga et al., Prostate 15; 75(3):242-54, 2015). In addition, when combined with a PSMA ADC carrying an auristatin payload MMAE, mice with human PDX prostate tumors treated with enzalutamide showed increased tumor growth inhibition (DiPippo et al., Prostate 15; 76(3):325-34, 2016).

[0232] Most prostate cancer patients eventually progress to androgen deprivation therapy (ADT) resistant disease. The anti-PSMA ADCs disclosed herein (such as ARX517) inhibit tumor growth in enzalutamide-sensitive and resistant CDX and PDX prostate cancer models, and are therefore suitable for treating subjects with ADT resistant disease. ARX517 inhibits tumor growth in various enzalutamide-sensitive and resistant xenograft models and exhibits additive efficacy with enzalutamide in enzalutamide-sensitive tumor models.

[0233] Example It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes made thereto will inspire those skilled in the art and will be included within the spirit and scope of this application and the appended claims.

[0234] Example 1: Transient transfection Approximately 16 hours before transfection, CHO-S culture was inoculated at 0.75 × 10⁻⁶ mg / L. 6 / mL was seeded in FreeStyle CHO medium. The next day, when the cell count reached 1.4 × 10⁹ / mL... 6 / mL to 1.6×10 6 Transfect cells at a concentration of / mL. At target cell count, add 400mM p-acetylphenylalanine stock solution to a final culture concentration of 1.4mM.

[0235] Polyethyleneimine / DNA (PEI / DNA) complexes were prepared as follows: DNA (1.42 ug / 1×10⁻⁶) was added... 6 Dissolve 100 cells in RPMI medium (5% (v / v) of total culture volume), incubate the DNA / RPMI mixture at room temperature for 2 minutes, add PEI stock solution (1 mg / mL) to the DNA solution at a 3:1 ratio (PEI / DNA (ug / ug)), and incubate the mixture at room temperature for 5 minutes.

[0236] Gently add the mixture to the cell culture and vortex. Transfer the flask to a 32°C incubator. Perform Western blot analysis on day 6 post-transfection. Harvest the supernatant on day 7 post-transfection.

[0237] Example 2: Antibody Humanization Humanization of parental mouse anti-PSMA J591 monoclonal antibodies (mAbs) was achieved by transplanting mouse heavy and light chain CDRs (HC-CDR1:31-35, HC-CDR2:50-66, HC-CDR3:99-104 and LC-CDR1:24-34, LC-CDR2:50-56, LC-CDR3:89-97) onto different human frameworks selected for highest homology with the mouse framework sequence and cloned into the human IgG 1 / κ constant region as a backbone (Liu et al., Cancer Research, 57, 3629-36354, 1997). Multiple humanized mAb variants were generated using transient expression in HEK293 cells by pairing four human heavy chain (HC) variants with six light chain (LC) variants. Key reversion mutations were added to the LC framework region to preserve antibody binding activity. To select lead clones, the binding of mAb variant supernatant to PSMA-positive LNCaP cells was tested by flow cytometry. Table 3 describes the selected heavy chain variable region sequences and four light chain variable region sequences used for further studies. Binding analysis showed that the four humanized full-length variants (shown as humanized anti-PSMA variants 1, 2, 3, and 4 (Table 3)) retained binding affinity comparable to that of the chimera (Table 3), exhibiting binding affinity in the nanomolar range. As disclosed in Table 3, humanized anti-PSMA variant 1 comprises the heavy chain sequence of SEQ ID NO:8 and the light chain sequence of SEQ ID NO:9; humanized anti-PSMA variant 2 comprises the heavy chain sequence of SEQ ID NO:10 and the light chain sequence of SEQ ID NO:11; humanized anti-PSMA variant 3 comprises the heavy chain sequence of SEQ ID NO:12 and the light chain sequence of SEQ ID NO:13; and humanized anti-PSMA variant 4 comprises the heavy chain sequence of SEQ ID NO:14 and the light chain sequence of SEQ ID NO:15. The underlined alanine amino acids in the heavy chain sequences of SEQ ID NO: 8, 10, 12, 14 and 16 in Table 3 (“ A The instructions state that, according to the amino acid position 114 of the Kabat numbering scheme (see Kabat et al., NIH Publication No. 369-847, 1993), a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), should be incorporated at this position (see Example 3).

[0238] Table 3. Amino acid sequence of anti-PSMA antibody Example 3: Selection and Recombinant Expression of Anti-PSMA Antibodies A lead humanized anti-PSMA antibody (see Example 2) was selected and recombinantly expressed in CHO cells using both transient transfection (described in Example 1) and a stable pooling method to examine the expression of the humanized sequence. In both the transient transfection and stable pooling methods, proprietary techniques were constructed into the expression vector and CHO host cells, respectively (see, for example, WO2018 / 223108). In the PSMA antibody, the non-natural amino acid p-acetyl-L-phenylalanine (pAF) was incorporated at position A114 (Kabat numbering scheme (see Kabat et al., NIH Publication No. 369-847, 1993); Table 3 shows the expression of the humanized sequence). A The indicated position is the first amino acid residue in the constant region of the heavy chain.

[0239] The binding of candidate humanized J591 antibodies to human PSMA expressed on LNCaP cells was evaluated. The final selection of humanized J591 (huJ591) mAb candidates was based on the minimum number of reversion mutations required to successfully preserve and maintain binding affinity for human PSMA binding activity (data not shown).

[0240] Example 4: Purification of anti-PSMA antibody Humanized mAb variant 1 (Table 3) containing the non-natural amino acid pAF at Kabat position 114 in each heavy chain from clarified cell culture medium (HCCF) (hereinafter referred to as "ARX517 mAb" or "unconjugated mAb") was loaded onto a Protein A column (MabSelect SuRe, Cytiva) equilibrated at 25 mM sodium phosphate, 100 mM sodium chloride, pH 7.3. After loading, the column was washed with Wash Buffer I (20 mM sodium phosphate, 100 mM sodium chloride, pH 7.5) followed by Wash Buffer II (50 mM sodium acetate, pH 5.5) to remove host cell contaminants. ARX517 mAb was eluted from the column with Elution Buffer (50 mM sodium acetate, pH 3.4), combined, and the pH was adjusted to 5.5 with 1.0 M Tris base. ARX517 mAb was further purified by loading onto a cation exchange column (Capto SP Impres, Cytiva) equilibrated in 50 mM sodium acetate (pH 5.5). After loading, the column was washed with wash buffer (50 mM sodium acetate, pH 5.5), and the mAb was eluted from the column and pooled using a linear gradient of 20 column volumes to 100% elution buffer (50 mM sodium acetate, 0.5 M chloride, pH 5.5). The cation exchange cell buffer was exchanged to a reconstitution buffer (20 mM L-histidine, 2.5% trehalose, pH 6.0), concentrated to 40 mg / mL, filtered through a 0.22 μM filter, and stored at ≤-60 °C.

[0241] Example 5: Generation of Antibody-Drug Conjugates (ADCs) The purified ARX517 mAb, as described in Example 4, was conjugated with drug-adaptor compound 6 (AS269). Acetylhydrazine (Sigma) and AS269 were added to the ARX517 mAb in molar excesses of 300:1 and 10:1, respectively. The pH of the reaction mixture was adjusted to 4.0 with 2.0 M acetic acid, and then diluted with water to a final ARX517 mAb concentration of 30 mg / mL. The reaction mixture was incubated at 28 °C for 24 hours, and then the buffer was exchanged to a preparation buffer via ultrafiltration and percolation (UF / DF) to remove excess reagents. The resulting antibody-drug conjugate (hereinafter referred to as “ARX517 ADC” or simply “ARX517”) was filtered through a 0.22 μm filter and stored at -60 °C.

[0242] Example 6: Analysis and Characterization of ARX517 Peptide mapping Peptide maps of ARX517 mAb and ARX517ADC were obtained using reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS). Approximately 200 μg of sample was denatured in 6 M GdHCl (pH 8) with Tris buffer and reduced in 20 mM DTT by heating at 37 °C for 30 min. The sample was cooled to room temperature and the reduced thiol was alkylated by incubation in 45 mM iodoacetamide in the dark for 30 min. The sample buffer was then exchanged for trypsin digestion buffer containing 50 mM Tris-HCl (pH 8) using a 0.5 mL Amicon Ultra 10 kDa MWCO rotary filter. Sequencing-grade modified trypsin (Promega) was then added, and the sample was incubated at 37 °C for 4 h. To stop the reaction, 15 μL of 20% TFA was added to each sample. Subsequently, 20 μL of each sample was injected into a 2 × 250 mm Phenomenex Jupiter Proteo 90 Å (4 μm) column, and the peptides were separated using a gradient of TFA and acetonitrile at a flow rate of 0.3 mL / min. HCD tandem MS data were acquired using a Thermo Qexactive Plus mass spectrometer for the first 10 peaks of each MS scan. The raw data were analyzed using Thermo PepFinder software, and the peptide species were then manually verified. The mass spectrometry data confirmed that the AS269 conjugation occurred only at the heavy chain alanine 114 (“HA114”) site (Kabat number), where, in the ADC chromatogram, heavy chain peptides 101–119 shifted to the theoretical conjugation retention time (Figure 1A shows the peptide chromatograms of the unconjugated mAb and ARX517 ADC detected by reversed-phase HPLC with mass spectrometry).

[0243] Hydrophobic interaction chromatography (HIC) HIC was used to determine the drug-to-antibody ratio (DAR) of ARX517 batches. Approximately 50 μg of each sample was loaded onto a MAbPac HIC-butyl HPLC column (Thermo) and eluted at a gradient of ammonium sulfate in a phosphate buffer mobile phase at 0.5 mL / min. HIC method with UV detection at 280 nm resolved the unconjugated mAbs, DAR1, and DAR2 species of ARX517 (Figure 1B). DAR was calculated using the following equation: ; where d i It is the peak area of ​​the ADC with drug loading of i.

[0244] HIC assays confirmed that the mean drug-to-antibody ratio (DAR) of the ARX517 ADC was 1.9.

[0245] Size exclusion chromatography (SEC) Size variants in ARX517 were analyzed using an Agilent 1200 series HPLC system with UV detection at 280 nm via SEC on a TSKgel G3000SWXL column (Tosoh). Samples were injected at 50 μg loadings and separated by isocratic elution using a mobile phase of 200 mM potassium phosphate and 250 mM potassium chloride at pH 6.0, a flow rate of 0.5 mL / min, and a column temperature of 25 °C. The area percentages of high molecular weight molecular weight (HMW), monomers, and low molecular weight molecular weight (LWM) were calculated using Agilent ChemStation software. SE-HPLC data (Figure 1C) showed that the ARX517 ADC exhibited high purity (98% main peak) and minimal aggregation (1.7% high molecular weight components).

[0246] Differential scanning calorimetry (DSC) The thermal transition temperatures of ARX517 mAb and ARX517 ADC (diluted to 1 mg / mL with formulation buffer) were determined using a MicroCal capillary VP DSC. The sample and reference cells were loaded with sample and formulation buffer, respectively. The instrument was programmed to scan from 10 °C to 110 °C at a rate of 1 °C / min, with a data averaging period of 10 seconds and an equilibration time of 15 minutes. Buffer-to-buffer scans were recorded throughout the experimental sequence to obtain baseline scans for subtraction from the experimental data, ensuring the shape of the baseline scans was reproducible during the experiment. The raw data were processed using MicroCal PEAQ DSC software to calculate T0. m The DSC characterization showed that the conjugation had minimal effect on thermodynamic stability, with no detectable difference in the overall conformation between mAb and ADC batches. Figure 1D ).

[0247] Example 7: In vitro PSMA expression and ARX517 activity Following the manufacturer's recommendations, PSMA cell surface expression levels were quantified in various prostate cancer cell lines using the QiFi Kit (Agilent Dako, part number K007811-8). Each cell line was harvested using StemPro Accutase cell dissociation reagent (Gibco, catalog number A1110501) and incubated at 4°C for only 1 hour with mouse anti-human PSMA antibody (BioLegend, catalog number 342502, clone LNI-17), isotype control antibody (BioLegend, catalog number 401401, clone MGI-45), or FACS buffer. After washing, cells were incubated in the dark at 4°C for 45 minutes with FITC-conjugated anti-mouse secondary antibody, along with assembly beads and calibration beads. After washing and resuspending the cells and beads, FITC fluorescence was analyzed using FACSCanto II (BD Biosciences). PSMA receptor numbers were calculated using a batch-specific standard curve obtained from the calibration beads. PSMA expression ranges from <1,000 receptors / cell to 136,000 receptors / cell.

[0248] Prostate cancer cell lines including LNCaP (clone FGC, CRL-1740), MDA-PCa-2b (CRL-2422), 22Rv1 (CRL-2505), and PC-3 (CRL-1435) were purchased from ATCC and cultured in the recommended medium. C4-2 cells were purchased directly from MD Anderson Cancer Center and cultured in RPMI-1640 medium supplemented with 10% FBS and 100 U / mL penicillin / streptomycin. MDA-PCa-2b cells were initially propagated in male nu / nu mice (Charles River Laboratories), isolated, and then maintained in medium supplemented with 20% FBS, 100 U / mL penicillin / streptomycin, 25 ng / mL cholera toxin, 10 ng / mL mouse epidermal growth factor, and 5% 5% urea nitrogen. M ethanolamine phosphate, 100 pg / mL hydrocortisone, 45 nM sodium selenite and 5 In F-12K medium containing g / mL human recombinant insulin, as recommended by ATCC. All cell lines were used for in vitro assays within one month of thawing frozen vials. Cells were seeded at 3,000 cells / well in 96-well white plates using their respective media and incubated overnight at 37°C, 5% CO2. The next day, serially diluted ARX517 or MMAE was added to the cells, and the cells were incubated for 4 days at 37°C, 5% CO2. At the end of incubation, cell viability was measured using CellTiter-Glo 2.0 reagent (Promega) in a SpectraMax M5E spectrophotometer. IC50 was determined by 4 PL curve fitting using GraphPad Prism software (version 8.2.1). 50 The value was calculated by subtracting the activity % from 100% to obtain the E value for a 30 nM dose. max %.

[0249] Treatment of cell lines with ARX517 ADC in a 4-day cell proliferation assay resulted in effective sub-nanomolar activity (IC50) of ARX517 in cells expressing moderate to high PSMA levels (MDA-PCa-2b, LNCaP, and C4-2). 50 The cell lines exhibited low PSMA expression (≤0.5 nM) and high maximum efficacy. Minimal ARX517 activity was observed in cell lines with low or no PSMA expression (22Rv1 and PC-3), demonstrating the selectivity of ARX517, which requires PSMA expression to acquire activity. All tested cell lines were sensitive to auristatin, as the positive control MMAE strongly inhibited proliferation in a similar manner in all tested cell lines, with an IC50 value of ≤0.5 nM. 50 The values ​​ranged from 0.18 nM to 0.78 nM (see Figures 2A to 2E of the dose-response curves, which show relative cell viability (cell viability %) of untreated control samples), and Table 4 summarizes the PSMA expression and potency (IC50) of ARX517 ADC and MMAE in prostate cancer cell lines. 50 ) and maximum efficacy (E) max )).

[0250] Table 4. PSMA expression and in vitro cytotoxic activity of ARX517 and MMAE in human prostate cancer cell lines. The cytotoxic activity of the anti-PSMA ADC of this invention indicates a beneficial therapeutic effect on prostate cancer in a PSMA receptor copy number-dependent manner. PSMA receptor copy numbers are typically higher in cancerous prostate tissue compared to in cancer-free tissues. This differential expression of PSMA receptor copy numbers between cancerous and normal tissues can facilitate the reduction and / or elimination of target-related non-prostate tissue-related toxicity. This analysis shows that the ARX517 ADC can exhibit lower activity in tissues expressing low PSMA receptor copy numbers than in cancerous prostate tissues with high PSMA receptor copy numbers. Therefore, the background toxicity of the ARX517 ADC can be significantly reduced.

[0251] Example 8: Cross-reactivity binding of PSMA species via biolayer interference (BLI) Binding of ARX517 mAb or ARX517 ADC to PSMA / FOLH1 in humans, cynomolgus monkeys, and rats was measured using biolayer interferometry on an Octet RED96 system (Sartorius). An anti-human IgG Fc capture biosensor (Sartorius) was loaded with purified ARX517 mAb or ARX517 ADC in HBS-P+ buffer (Cytiva). Binding and dissociation kinetics of serially diluted PSMA / FOLH1 (human, cynomolgus monkey, or rat) in HBS-P+ buffer were monitored using either the ARX517 mAb-loaded biosensor or the ARX517 ADC-loaded biosensor. Reference data were obtained using parallel buffer blank subtraction. Affinity was calculated by globally fitting the processed binding curves using a Langmuir model describing a 1:1 binding stoichiometry.

[0252] The results are shown in Figure 3A to... Figure 3C As shown in Table 5, in the cross-species PSMA biolayer interference binding assay, ARX517 mAb (unconjugated) showed high affinity binding similar to human and cynomolgus monkey PSMA (apparent KD values ​​of 0.62 nM and 0.79 nM, respectively; Figures 3A and 3B), and did not bind to rat PSMA ( Figure 3C The ARX517 ADC binds to human, cynomolgus monkey, and rat PSMA with similar affinity to the unconjugated ARX517 mAb, thus confirming that the conjugation of AS269 at site HA114 does not affect the binding activity of the ADC. Figure 3A- Figure 3C ).

[0253] Table 5. Dissociation kinetics of huJ591 mAb and ARX517 on PSMA in humans, rats, or cynomolgus monkeys. .

[0254] Example 9: Pharmacokinetics and stability of ARX517 after administration in mice with and without C4-2 tumors Sexual research To assess the in vivo stability and pharmacokinetics of ARX517, male nude (nu / nu) mice with or without C4-2 prostate tumors were administered a single intravenous dose of ARX517 at 1 mg / kg or 5 mg / kg (n=5 / group). Blood samples were collected from all animals before administration and up to 28 days after administration (0.5 h, 2 h, 6 h, 24 h, 48 h, 72 h, 168 h, 240 h, 336 h, 504 h, and 672 h post-administration). Blood samples were immediately diluted 10-fold in casein-PBS (Thermo Scientific catalog number 37528) and frozen in tubes at -60°C to -80°C. Samples were analyzed in qualified ligand binding assays designed to measure total antibody (“TA”; unconjugated and conjugated antibody species) and intact ADC (ARX517 ADC DAR2 species only, i.e., 2-drug ADC species only), as described below.

[0255] Bioanalytical assays in mouse matrix: The ARX517 TA assay was developed to detect unconjugated and all conjugated antibody species in naked (nu / nu) mouse serum. Meso Scale Discovery (MSD) high-binding plates were coated overnight at 4°C with recombinant human PSMA (rhPSMA, R&D Systems catalog 4234-ZN-010), followed by blocking the next day at room temperature with casein-PBS (Thermo Scientific catalog 37528) for at least 1 hour. The plates were washed three times with 1x wash buffer (20x KPL wash solution, KPL catalog 50-63-04), and duplicate copies of the ARX517 standard (STD), quality control (QC), and a 1:50 pre-diluted sample in casein-PBS were added to the plates. After incubation at room temperature for 2 hours, the MSD plates were washed three times as previously described, and biotinylated goat anti-human κ detection antibody (Southern Biotech catalog 2061-08) was incubated at room temperature for 1 hour. Following a washing step to remove unbound antibodies, streptavidin-SULFO-tagged reagent (MSD catalog number R32AD-1) was added at room temperature and allowed to stand for 1 hour. After a final washing step, 1x read buffer T (4x read buffer T, MSD catalog number R92TC-2) was added, and the plate was read in an MSD QuickPlex SQ120 unit. The LLOQ measured in nu / nu mouse serum was 39.1 ng / mL. The ARX517 intact ADC assay in null (nu / nu) mouse serum was designed to specifically detect only ADCs with two drug linkers (not ADCs with one drug linker, unconjugated mAbs, or free pAF-AS269) to allow for the identification of any loss of AS269 drug linkers when comparing intact ARX517 ADC curves with TA curves. The assay procedure was similar to the TA assay, except that the MSD high-binding plate was coated with AMB-20 (an anti-AS269 specific rabbit monoclonal Ab (mAb)) and the detection antibody was biotinylated AMB-20. The LLOQ for complete ADC assay in nu / nu mouse serum was 125 ng / mL. Pharmacokinetic (PK) parameters of the TA and complete ADC data were analyzed in PhoenixWinNonlin software using non-compartmental analysis and linear upper / log lower trapezoidal methods. At least three time points were required to determine the elimination rate constant, and parameters were set based on this calculation. Concentrations below the limit of quantitation (BLQ) were set to zero for PK analysis.

[0256] Comparison of the TA and intact ADC concentration-time curves allowed for rigorous detection of any loss of AS269. ARX517 exhibited overlapping TA and intact ADC concentration-time curves at both dose levels, indicating no observed deconjugation of the drug-connector, and ARX517 was stable in cycles during the 28-day study period (Figure 4A).

[0257] Linear pharmacokinetic (pK) and prolonged stability were observed after administration of ARX517 to mice with and without C4-2 tumors (Table 6). Mean pK parameters were analyzed using non-compartmental analysis (n = 5 mice / group). The complete ARX517 ADC was comparable to TA exposure at both doses, with the mean area under the curve (AUC) from time zero to the final quantifiable concentration being [data missing]. 0-最后 The ratio was 1.07 to 1.23. In tumor-free mice (mean t... 1 / 2 The range was 261 to 350 hours) and in mice carrying tumors (mean t 1 / 2 The range is 210 hours to 237 hours), resulting in the terminal half-life (t) of the ADC. 1 / 2 The t value is relatively long. For tumor-bearing mice, t 1 / 2 It appears slightly shorter, but the differences may fall within the variability of the study. Mean AUC of ADC in tumor-bearing mice compared to non-tumor-bearing mice. 0-最后 The ratios were comparable, ranging from 0.86 (1 mg / kg) to 1.03 (5 mg / kg), indicating that there was no significant target-mediated drug treatment in the C4-2 xenograft model. Figure 4B ).

[0258] Table 6. PK parameters after ARX517 administration in mice with and without CR-2 tumors. .

[0259] Example 10: Stability of ARX517 in human serum ARX517 was diluted to 200 μg / mL in pooled human serum (Bioreclamation catalog number HMNSRM). Aliquots (100 μL) in sterile 0.6 mL Eppendorf tubes were incubated upright at 37°C with 5% CO2 for 21 days. Individual tubes were removed after incubation at days 0, 1, 4, 6, 8, 11, 14, and 21 and frozen at -80°C until all time points were collected. All samples were diluted to 500 ng / mL in human serum to fall within the quantitative range of the PK assay and analyzed in duplicate using the MSD platform for total antibody (TA) and intact ADC assays to assess stability. The TA and intact ADC assays in human serum were captured and detected using the same reagents as those used for the mouse TA and intact ADC assays (see Example 9). The LLOQs for the TA and intact ADC assays in human serum were 65.3 ng / mL and 32.7 ng / mL, respectively. The concentrations of TA and intact ADC were highly similar at all time points, with the ADC-to-TA ratio ranging from 0.91 to 1.16 (Table 7). The percentage differences between TA and intact ADC and theoretical (PDT) values ​​were both within 30%, which is considered within assay variability. Overall, the data demonstrate that ARX517 is stable in human serum for 21 days under the assay conditions tested.

[0260] Table 7. Stability of ARX517 in human serum .

[0261] Example 11: In vivo efficacy of ARX517 in mouse CDX and PDX models ARX517 was evaluated in a range of in vivo cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

[0262] Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models: C4-2 and MDA-PCa-2b cells were purchased from ATCC (CRL-1595 and CRL-2422, respectively) and grown to near confluence. Freshly harvested cells were suspended in PBS and mixed 1:1 with matrix gel. NCG or nu / nu mice were anesthetized with isoflurane (2% to 3%, 2 L / min oxygen) and 5 × 10⁻⁶ cells were implanted subcutaneously on the right side. 6 0.2 mL of cell suspension per mouse. NSG mice containing subcutaneously implanted TM00298 PDX cells were obtained from Jackson Laboratories. For the CTG-2440 study (ChampionsOncology), NOG mice were subcutaneously implanted with cells derived from 1000 mmHg cells. 3 Up to 1500mm 3Fragments of tumors from donor mice.

[0263] Twice a week, weight was recorded, and both tumor length and width were measured using electronic calipers. Tumor volume (TV) was calculated as L x W x W x 0.5 (where L is tumor length and W is tumor width). When the tumor volume equals 150 mm... 3 Up to 500mm 3 Mice were randomly assigned to approximately equal TV mean groups and administered the mediator or the designated test product intravenously at 10 mL / kg. Enzalutamide was administered orally at 10 mg / kg in 1% carboxymethyl cellulose and 0.1% Tween-80. The percentage of tumor growth inhibition (TGI%) was calculated as follows: 100 × (1 - [tumor volume or wet weight of the treatment group / tumor volume or wet weight of the control group]).

[0264] In these studies, ARX517 did not induce severe weight loss, mortality, or morbidity.

[0265] Enzalutamide-sensitive MDA-PCa-2b tumor model.

[0266] MDA-Pca-2b cells were subcutaneously implanted into the lateral ventral region of male nude mice (n=10 / group). When the tumor reached 100mm... 3 Up to 200mm 3 At that time, with Figure 5 The doses shown represent a single intravenous dose of the test product administered to mice (dashed line, day 14). TGI% was calculated based on tumor wet weight on day 45 post-cell implantation. Statistical analysis: For tumor wet weight on day 45, * = p < 0.05 and ** = p < 0.01, calculated using the nonparametric Mann-Whitney t-test. A single intravenous administration of ARX517 demonstrated a robust TGI of up to 83% (at doses of 5 mg / kg and 10 mg / kg); however, 10 mg / kg of unconjugated mAb or allotype control ADC (containing AS269) did not inhibit tumor growth. Figure 5 The figure shows the mean tumor volume ± SEM over time.

[0267] Enzalutamide-sensitive TM00298 prostate cancer PDX model.

[0268] NSG mice were subcutaneously implanted with tumor cells derived from the TM00298 patient. When the tumor reached 100 mm... 3 Up to 200mm 3Mice were administered the test product intravenously once weekly (as shown in Figure 6A; dashed lines indicate the ARX517 dose). TGI% was calculated based on tumor volume measurements at the end of the study. Statistical analysis: * = p < 0.05, ** = p < 0.01 and *** = p < 0.001; nonparametric Mann-Whitney t-test and final tumor volume calculations were used. Weekly ARX517 administration inhibited tumor growth in a dose-dependent manner (up to 66% in the 3 mg / kg group, p < 0.0001, relative to the medium), and daily enzalutamide administration (10 mg / kg) induced 41% TGI (p < 0.01). In this TM00298 model, the combination of ARX517 and enzalutamide resulted in 85% TGI (p < 0.0001) (Figure 6A; figure shows mean tumor volume ± SEM over time).

[0269] CTG-2440 PDX prostate cancer model.

[0270] NOG mice (n=10 / group) were subcutaneously implanted with 1000mm... 3 Up to 1500mm 3 CTG-2440 tumor fragments were stored in mice. The test product was administered intravenously to mice once weekly. Figure 6B (Dash line indicates ARX517 dose). TGI% was calculated based on tumor volume measurements at the end of the study. Statistical analysis: * = p < 0.05, ** = p < 0.01, and *** = p < 0.001; calculated using one-way ANOVA followed by Tukey's multiple comparison test. Weekly administration of ARX517 as a single agent inhibited tumor growth by up to 92% in a dose-dependent manner. In this model, at 3 mg / kg, ARX517 induced a more robust TGI than enzalutamide at 10 mg / kg (92% vs. 38%, respectively). Figure 6B The figure shows the mean tumor volume ± SEM over time.

[0271] Most prostate cancer patients eventually progress to androgen deprivation therapy (ADT) resistant disease. ARX517 was evaluated in an enzalutamide-resistant C4-2 CDX model. Nude mice (n=5-10 / group) were subcutaneously implanted with C4-2 tumor cells in streptomycin. When the tumor reached 200 mm... 3 Up to 500mm 3 At that time, as shown in Figure 7A and Figure 7BThe test product was administered intravenously to mice at the indicated dose (dashed lines indicate ARX517 dose; the figure shows mean tumor volume ± SEM over time). TGI% was calculated based on tumor volume measurements at the end of the study. Statistical analysis: **=p<0.01 and ***=p<0.001, calculated using two-way ANOVA with repeated measures and Tukey post-hoc test. ARX517 promoted C4-2 TGI after single doses as low as 1 mg / kg and observed no disease progression after a single dose of 5.0 mg / kg (Figure 7A). In the C4-2 model, weekly administration of ARX517 resulted in TGIs ranging from 37% (1 mg / kg) to 79% (3 mg / kg). Consistent with C4-2 enzalutamide resistance, the combination of ARX517 and enzalutamide (10 mg / kg) did not result in an additive TGI. Figure 7B ).

[0272] Example 12: Pharmacokinetic and Toxicological Studies of ARX517 in Rats and Cynomolgus Monkeys Rat Studies. ARX517 was evaluated in a single-dose Good Laboratory Practice (GLP) toxicity study in Sprague-Dawley rats. Male and female Sprague-Dawley rats (7 to 10 weeks old) were administered the carrier or ARX517 (20 mg / kg, 40 mg / kg, or 60 mg / kg) via intravenous infusion over approximately 20 minutes at a dose volume of 15 mL / kg. Rats were observed for 28 days following administration of the test product, and necropsy was performed on day 29. Toxicological observations / evaluations were performed using standard methods, including: mortality, clinical observation, injection site observation, body weight, food consumption, safety pharmacology tests (functional observation kit and respiratory examination), clinicopathology (hematology, coagulation, serology, and urinalysis), toxicokinetics, gross observation, terminal necropsy organ weight, and histopathological assessment. Jugular venous blood samples were collected at different time points after administration for clinicopathological and toxicokinetic analysis. For histopathology, all major tissues were trimmed and fixed in 10% neutral buffered formalin, except for the eyes, testes, and epididymis containing the optic nerve, which were fixed in a modified Davidson solution for 24 to 72 hours. The preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under a microscope by a licensed pathologist.

[0273] Because ARX517 does not bind to rodent PSMA (see Example 8, Table 5), this rat study revealed the acute target-dependent toxicological characteristics of ARX517. Single administration of a dose of ARX517 to rats via intravenous infusion over 20 minutes at doses of 20 mg / kg, 40 mg / kg, or 60 mg / kg did not result in treatment-related mortality, near-death experiences, abnormal behavioral tests, or respiratory findings. On day 4 post-infusion, clinicopathological findings included increased neutrophil and monocyte counts, decreased lymphocyte counts, reduced erythrocyte parameters (RBC / HGB / HCT), and changes in liver function (increased AST / ALT / ALP / TBIL) and kidney function (increased CRE) at doses ≥40 mg / kg. Significant test-product-related weight increases were observed in the liver, spleen, and lungs in both sexes. At the end of the 28-day observation period, adverse histopathological findings related to ARX517 were observed in male reproductive organs and lungs of both sexes at doses ≥20 mg / kg, and the maximum tolerated dose (MTD) of ARX517 was 60 mg / kg.

[0274] The cynomolgus monkey study evaluated ARX517 in a repeated-dose GLP toxicity study in cynomolgus monkeys. Male and female cynomolgus monkeys (2.5 to 3.5 years old, n=6 / sex / group) were administered the carrier or ARX517 (1 mg / kg, 6 mg / kg, or 9 mg / kg) twice, 3 weeks apart, via intravenous infusion over approximately 20 minutes. One week after the final ARX517 dose, four monkeys / sex in each group were euthanized and autopsied, and the remaining two animals / sex (the recovery group) were observed for another six weeks, followed by a final autopsy. Toxicological observations / evaluations were performed using standard methods, including: mortality, clinical observation, injection site observation, body weight, food consumption, safety pharmacology tests (functional observation kit and respiratory examination), clinicopathology (hematology, coagulation, serology, and urinalysis), toxicokinetics, gross observation, final autopsy organ weight, and histopathological evaluation. Jugular venous blood samples were collected at different time points after administration for clinicopathological and toxicokinetic analysis. For histopathology, all major tissues were trimmed and fixed in 10% neutral buffered formalin, except for the eyes, testes, and epididymis, which were fixed in a modified Davidson solution for 24 to 72 hours. The preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined under a microscope by a licensed pathologist.

[0275] To determine the serum concentrations of total ARX517 antibody (A), ARX517 ADC, and free payload pAF-AS269, blood samples were collected from all available study animals (n=6 / sex / group) at 0 h (pre-dose), 0.5 h, 4 h, 8 h, 24 h, 72 h, 120 h, 168 h, 336 h, and 504 h after the first ARX517 dose, and at 0.5 h, 4 h, 8 h, 24 h, 72 h, 120 h, and 168 h (dose phase), and at 336 h, 504 h, 672 h, 840 h, and 1176 h (recovery phase) after the second ARX517 dose. Blood samples were collected into tubes without anticoagulant, allowed to coagulate at room temperature for at least 30 min, and centrifuged at 3200 × g for 15 min at 4 °C. Transfer the serum to aliquots and store in a refrigerator set to ≤-65°C.

[0276] Samples were analyzed in a validated PK assay. The TA PK assay in cynomolgus monkey serum used the same capture and detection reagents as the TA assay in mouse matrix (see Example 9). The ARX517 ADC PK assay in cynomolgus monkey serum was similar to the complete ADC assay in mouse matrix (Example 9), except that the MSD high-binding plate was coated with rhPSMA. The limits of quantitation (LLOQ) for TA and ADC assays in cynomolgus monkey serum were 78.1 ng / mL and 19.5 ng / mL, respectively.

[0277] To extract pAF-AS269 from 30 μL of research sample, STD, and QC, a protein precipitation step was performed in a 96-well deep plate using a 200 ng / mL internal standard (diclofenac) and 190 μL of 0.1% formic acid in acetonitrile. The sample, STD, and QC were vortexed for 10 min, centrifuged at 3220 × g for 15 min at 4 °C, and 100 μL of the supernatant was transferred to a new 96-well deep plate. 100 μL of deionized water was added to the transferred supernatant, vortexed for 5 min, and centrifuged at 3220 g for 5 min at 4 °C. 10 μL of the extracted sample was injected into LC-MS / MS for chromatographic separation and detected using cation ESI mode. The LLOQ for pAF-AS269 in cynomolgus monkey serum was 0.2 ng / mL.

[0278] Because ARX517 binds to cynomolgus monkey PSMA and human PSMA with similar affinity (see Examples 8 and Table 5), the target and off-target toxicological characteristics of ARX517 were evaluated in cynomolgus monkeys following two intravenous administrations at 3-week intervals of 1 mg / kg, 6 mg / kg, or 9 mg / kg. In this study, the no obvious adverse effect level (NOAEL) of ARX517 was 1 mg / kg / dose for both male and female monkeys, and the highest non-serious toxic dose (HNSTD) was 6 mg / kg / dose. On day 6 after the second ARX517 administration, clinicopathological findings were all considered harmless, observed only at doses ≥6 mg / kg, and included: increased monocyte count, decreased platelet (PLT) count, increased clotting time (APTT), and increased hepatocyte enzymes (AST / ALP / GGT). Under HNSTD, although histopathological target organs included the liver, spleen, and thymus, all findings of damage were normalized, and the incidence / severity decreased at the end of the 6-week recovery period. At a dose of 9 mg / kg (higher than HNSTD), additional target organs were the kidneys and lungs. No ophthalmic, respiratory, urinary, cardiovascular, or neurological findings were observed at any dose.

[0279] ARX517 toxicokinetics in repeated-dose studies in cynomolgus monkeys demonstrate the stability of ARX517 in systemic circulation, with overlapping TA and ARX517 ADC concentration-time curves at all doses (Figure 8A; showing mean ± SD from n=12 monkeys / group). TA exposure was similar to ADC exposure, with mean maximum observed concentration (C0). max ) and AUC 最后 The ratio is between 0.77 and 1.07 (Table 8). Table 8: ARX517 toxicokinetic parameters in repeated-dose toxicity studies in cynomolgus monkeys Following administration of a second dose of ARX517, the free payload pAF-AS269 released from ARX517 was non-quantitative for all animals in the 1 mg / kg dose group and 3 animals in the 6 mg / kg dose group. The remaining animals in the 6 mg / kg or 9 mg / kg dose groups showed a slow emergence of pAF-AS269 in circulation, with a few quantifiable time points at very low concentrations (<0.5 ng / mL) close to the LLOQ assay (Figure 8; showing mean ± SD of pAF-AS269). These low concentrations of pAF-AS269 were >200-fold lower (IC50-fold) than the observed in vitro activity of pAF-AS269. 50 >100 nM or 111 ng / mL; data not shown). Internalization and intracellular proteolytic degradation of ARX517 result in the release of ARX517 as a free payload (or metabolite; see [link to relevant documentation]). Figure 8C The structure of pAF-AS269 is essential for its formation and is associated with the slow appearance of free pAF-AS269 in circulation (observed 96 to 120 hours after ARX517 administration). max ).

[0280] In male monkeys, the ARX517 AUC after a second dose was 6 mg / kg HNSTD. 最后 C max and t 1 / 2 The values ​​were 36,500,000 h*ng / mL, 175,000 ng / mL, and 324 hours (approximately 13.5 days), respectively (Table 8). In C4-2 tumor-bearing mice, at a pharmacologically active dose of 5 mg / kg (see Example 9), the mean AUC of ARX517 was... 最后 C max and t 1 / 2 The values ​​were 7,570,000 h*ng / mL, 57,100 ng / mL, and 237 hours, respectively (Table 6 and...). Figure 4B The AUC of ARX517 exposure at a dose of 5 mg / kg, evaluated in the PK study (higher than the pharmacologically active dose of 3 mg / kg observed in multiple models), compared to the exposure at HNSTD in the monkey toxicology study, is shown. 最后 More than 5 times and C max More than 3 times the significant therapeutic index ( Figure 9 ).

[0281] In summary, pharmacological, toxicological, and pharmacokinetic characteristics indicate that ARX517 is well tolerable at exposures much higher than those required for efficacy, thus providing a theoretical basis for clinical investigation as a potential treatment option for mCRPC.

[0282] Example 13: A phase 1 / 2, multicenter, open-label, dose-escalation and dose-expansion study to evaluate ARX517. Safety, pharmacokinetics and antitumor activity The treatment options for subjects with metastatic castration-refractory prostate cancer that were resistant or refractory to previous standard therapy were randomized.

[0283] ARX517 ( Figure 10 It is currently being evaluated in a multicenter phase 1 clinical trial in the United States (ARX517-2011 (APEX-01); NCT04662580 (see, on the World Wide Web, In short, the APEX-01 trial was a phase 1 / 2, multicenter, open-label, dose-escalation and dose-expansion study to evaluate the safety, pharmacokinetics (PK), and antitumor activity of ARX517 in subjects with metastatic castration-refractory prostate cancer (mCRPC) that were resistant or refractory to prior standard therapy, in a randomized comparison with investigator's choice of treatment (ICT).

[0284] This first-in-human phase 1 / 2 multicenter open-label study aims to evaluate the safety, pharmacokinetics, pharmacodynamics (PDy), and preliminary antitumor activity of ARX517 in subjects with mCRPC who are resistant or refractory to standard therapy. Phase 1a and 1b dose-escalation and dose-expansion phases are designed to identify the MTD and / or RP2D. In phase 2, subjects will be randomized to receive ARX517 at either RP2D or ICT as a comparator. The ICT to be used in phase 2 will be determined after reviewing all available clinical data from phase 1.

[0285] Primary objectives: safety, tolerability, maximum tolerated dose (MTD), and recommended phase 2 dose (RP2D).

[0286] Secondary targets: PK, immunogenicity, preliminary antitumor activity (RECIST 1.1), prostate-specific antigen (PSA) response [PSA30, PSA50, PSA90], PCWG3.

[0287] Key eligibility criteria. Key inclusion criteria included: male subjects ≥18 years of age at the time of initial written informed consent; histologically confirmed prostate cancer; documented metastatic disease; castration-refractory prostate cancer according to Prostate Cancer Working Group 3 (PCWG3); ongoing therapy with gonadotropin-releasing hormone agonists or antagonists (unless previously orchiectomized) (and willing to continue), and serum testosterone levels <50 ng / dL at screening; prior treatment for metastatic prostate cancer with at least two FDA-approved therapies, at least one of which is an androgen receptor signaling inhibitor; adequate blood cell count; disease progression due to PSA, RECIST 1.1, or new bone lesions; Eastern Cooperative Oncology Group (ECOG) performance status ≤ 1.

[0288] Key exclusion criteria. Key exclusion criteria include: subjects with central nervous system (CNS) metastases unless the CNS metastases have been treated with topical therapy and have been demonstrated to be stable in the last 2 months prior to the enrollment date and do not currently require ongoing systemic steroid therapy; a history of any aggressive malignancy (other than primary) requiring active treatment in the 2 years prior to the enrollment date; a significant baseline prolongation of the QT / QT interval corrected for heart rate (QTc) using the Fridricia QT correction formula, e.g., a triplicate mean QTc interval >480 ms (CTCAE grade 1); a prior history of interstitial lung disease, pneumonia, or other clinically significant lung disease in the 12 months prior to the enrollment date; a clinically significant ocular finding by a qualified ophthalmologist or optometrist, including active ocular infection or chronic corneal disease; and continued use of bisphosphonates or denosumab therapy, estrogen therapy, systemic glucocorticoids (>10 mg prednisone), or finasteride / dutasteride.

[0289] Design Details. Primary Objective: Treatment. Intervention Model: Single-group allocation. Intervention Model Description: This is the first phase 1 / 2 multicenter open-label study in humans to evaluate the safety, pharmacokinetic (PK), PDy, and preliminary antitumor activity of ARX517 in subjects with mCRPC who are resistant or refractory to standard therapy. Phase 1a and 1b dose-escalation and dose-expansion phases will identify the MTD and / or RP2D. Phase 2 will randomize subjects to receive ARX517 at either RP2D or ICT as a comparator. The ICT to be used in Phase 2 will be determined after reviewing all available clinical data from Phase 1. Masking: None (open-label).

[0290] Participant Groups / Groups: Experiment: ARX517 (Phase 1 Dose Escalation and Expansion). During the dose escalation phase of the study, subjects were recruited into a group that received escalating dose levels of ARX517 via intravenous (IV) infusion every 3, 4, or 6 weeks. During the dose expansion phase of the study, subjects received dose levels and intervals at or below the mean time to dose (MTD), which were identified as putative RP2D.

[0291] Intervention / treatment: Drug: ARX517. ARX517 is an ADC consisting of two (2) humanized anti-PSMA monoclonal antibodies (mAbs) (IgG1κ) covalently conjugated to two proprietary microtubule-disrupting toxins called AS269.

[0292] Primary outcome measures. Phase 1: Assess the incidence of adverse events. The incidence and severity of adverse events or serious adverse events of ARX517 were assessed using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events, Version 5 (CTCAE) (time range: 1.5 years) to determine the safety and tolerability of the treatment.

[0293] Secondary outcome measures. (A) Phase 1: Area under the serum concentration-time curve (AUC) of ARX517. The area under the pharmacokinetic parameters (AUC) of the serum concentration-time curve will be analyzed using different analytes (such as ADC, total antibody, and pAF-AS269) (time range: 3 years). (B) Phase 1: Maximum serum concentration (Cmax) of ARX517. The maximum serum concentration (Cmax) of the pharmacokinetic parameters will be analyzed using different analytes (such as ADC, total antibody, and pAF-AS269) (time range: 3 years). (C) Phase 1: Trough concentration (Cmax) of ARX517. 谷 The pharmacokinetic parameter trough concentration (C) will be analyzed using different analytes, such as ADC, total antibody, and pAF-AS269. 谷 (D) Phase 1: Incidence of ADA anti-ARX517. Assess the incidence of anti-drug antibody (ADA) anti-ARX517 at selected time points (time range: 3 years). (E) Overall Survival. Overall survival (OS) is defined as the time from the first dose of the study therapy to the date of death (any cause). Review surviving subjects at the last known time of their survival (time range: 3 years). (F) Assess changes in serum prostate-specific antigen (PSA) levels. The proportion of subjects showing a confirmed decrease in serum prostate-specific antigen (PSA) levels (PSA30, PSA50; time range: 3 years) from baseline of 30% and 50%. (G) Progression-Free Survival (PFS). PFS is defined as the time between the date of the first dose of the study therapy and the date of progression or death (whichever occurs first), calculated for subjects with an evaluable response. Review subjects at subsequent therapy (time range: 3 years).

[0294] Other outcome measures. (A) Phase 1: Assessment of biomarkers. Exploratory blood and tissue-based biomarkers related to response to the investigational drug were assessed (time range: 3 years). (B) Phase 1: Assessment of PSMA expression. Baseline PSMA expression was assessed (time range: 3 years). (c) Phase 1: Assessment of changes in the Brief Pain Scale. The Brief Pain Scale questionnaire was used to assess the subjects' quality of life. Higher scores indicated poorer outcomes (time range: 3 years). (D) Phase 1: Assessment of changes in the Functional Assessment of Cancer Therapy-P (FACIT-P) in patients with prostate cancer. The FACIT-P questionnaire was used to assess the subjects' quality of life. Higher scores indicated poorer outcomes (time range: 3 years).

[0295] Study design. The study consists of two parts: a dose escalation part and a dose amplification part.

[0296] Dose escalation. The dose escalation portion of the study consisted of escalating dose levels of ARX517 administered as a single agent to determine MTD and RP2D. Subjects will be recruited using an i3+3 design (Liu et al., 2019, J BiopharmStat. 2019:1-11), with a starting dose of 0.32 mg / kg.

[0297] Dose expansion. The dose expansion portion of the study will assess the putative RP2D. Patients for dose escalation will be recruited using an i3+3 design. If no DLT is observed during the 21-day DLT period, subsequent dose cohorts may be recruited after the Safety Monitoring Committee (SMC) reviews available safety and tolerability data. The study begins with the following: evaluation of 6 dose levels of ARX517 Q3W according to the modified Fibonacci protocol as shown in Table 9.

[0298] Table 9. ARX517 Q3W Dosage Levels According to the Modified Fibonacci Regimen .

[0299] Additional groups were added after the start of the clinical trial, with dose level increments ≤ 20% of the highest dose level in the subsequent groups, as recommended by the SMC. This study recruited patients in cohort 7 (2.4 mg / kg, Q3W), cohort 8 (2.88 mg / kg, Q3W), cohort 9 (3.4 mg / kg, Q3W), and cohort 10 (4.5 mg / kg, Q3W). Figure 12 The data presented in this paper was available through queue 8 at the time of submission of this application.

[0300] Preliminary results from patients in cohorts 1 through 6 of the APEX-01 clinical trial. In the phase 1 dose-escalation portion of the study, ARX517 was administered as a single-dose agent at escalating dose levels every 3 weeks. The primary endpoints were safety, tolerability, and pharmacokinetics. Key secondary endpoints were an objective decrease in prostate-specific antigen (PSA) from baseline and / or tumor shrinkage. PSA is a protein produced by the prostate gland and is commonly used as a biomarker for diagnosing and tracking prostate cancer. A ≥50% reduction in PSA levels from baseline was considered clinically relevant and has been shown to be associated with improved overall survival in prostate cancer. In prostate cancer patients, at a dose level of 2.0 mg / kg, a PSA level reduction greater than 50% was observed in three out of three patients. Two patients in this cohort experienced a PSA reduction >90%. Furthermore, one of these three patients had measurable soft tissue disease and experienced a partial response on the scan at the time of the first treatment.

[0301] Results from patients in cohorts 1 through 8 of the APEX-01 clinical trial. An i3+3 dose-escalation design was used. Eligible patients had ≥2 prior FDA-approved treatments for mCRPC in which progression was achieved according to the Prostate Cancer Working Group criteria. Key objectives included safety, pharmacokinetics (PK), and clinical efficacy. Baseline PSMA PET expression was not required for eligibility but was assessed as a biomarker.

[0302] Twenty-four (24) patients received ARX517 Q3W via intravenous infusion at escalating doses (Table 10). Patients had a median of four prior therapies; 100% received ≥1 androgen pathway inhibitor, 50% received taxanes, and 12.5% ​​received PSMA-targeted radionuclides. Grade 1 / 2 treatment-related adverse events (TRAEs) were dry mouth (41.7%), fatigue (33.3%), diarrhea, and thrombocytopenia (20.8% each). Four Grade 3 TRAEs were reported at 1.7 mg / kg, 2.4 mg / kg, and 2.88 mg / kg (both with lymphopenia and thrombocytopenia). No dose-limiting toxicities (DLTs), treatment-related serious adverse events (SAEs), or ≥Grade 4 adverse events were reported. At higher doses (cohorts 4 to 8), the median duration of treatment was 6.3 months (range 0.7+, 11.8+). In addition, a decrease of >50% in PSA and circulating tumor DNA (ctDNA) was observed (Table 10); RECIST v1.1 response was confirmed in 2 out of 7 patients. The p-p ...

[0303] Table 10. Assessment of PSA and ctDNA decrease, confirmed response, DLT, and grade 3 / 4 / 5 according to ARX517 dose levels. TRAE .

[0304] *n / d = No data available.

[0305] During treatment, changes in ctDNA have been shown to predict time to progression and survival (see Tolmeijer SH et al., Clin Cancer Res. 2023 Aug 1; 29(15):2835-2844, doi: 10.1158 / 1078-0432.CCR-22-2998; Sartor O. Clin Cancer Res. 2023 Aug 1; 29(15):2745-2747, doi: 10.1158 / 1078-0432.CCR-23-1043). Serial plasma samples were collected at baseline, C3D1, C4D1, and EOT, showing the best percentage change from baseline. ctDNA was measured using the GuardantINFINITY assay (Guardant Health), which showed a specificity of 96.9%, a sensitivity of 91.3%, and a reported limit of detection of 0.06%. Samples were processed after passing through multiple quality control measurements, including DNA yield, GC bias, methylation bias, diversity, and contamination checks. Changes in ctDNA compared to baseline levels were measured based on aggregated tumor-specific methylation signal fractions. A reduction in ctDNA greater than 50% was observed in 81% (17 / 21) of patients (cohorts 4–8); see [link to relevant documentation]. Figure 19 .

[0306] ARX517 treatment resulted in a decrease in PSA and a RECIST v1.1 response, but no treatment-related SAEs. Dose expansion to 3.4 mg / kg (cohort 9) and 4.5 mg / kg (cohort 10) has begun. Figure 12 (Queue 10 is not shown in the diagram).

[0307] Pharmacokinetic results of patients in cohorts 1 through 7 of the APEX-01 clinical trial.

[0308] As disclosed herein, ARX517 is an anti-PSMA ADC containing an anti-PSMA mAb conjugated to AS269 at a drug-to-antibody ratio (DAR) of 2 (2). The ARX517 design addresses the instability issues encountered by previous PSMA-targeted ADCs through three key components: a non-cleavable PEG linker, a non-cellularly permeable payload, and stable oxime conjugation chemistry. Site-specific conjugation of AS269 to the mAb is achieved using synthetic amino acid (pAF) incorporation. This ADC design minimizes premature release of the free payload and off-target toxicity.

[0309] Twenty-one (21) patients in cohorts 1 through 7 received ARX517 Q3W via intravenous infusion at doses ranging from 0.32 mg / kg to 2.4 mg / kg. Serum samples were collected from patients at fixed time points and evaluated in validated total antibody (TA; the sum of unconjugated and conjugated antibodies), ADC (conjugated antibodies with DAR of 1 or 2), and free payload pAF-AS269 assays. The assays for ARX517 TA, ADC, and pAF-AS269 PK in patient serum samples were similar to those for TA and intact ADC in mouse serum (see Example 9) and pAF-AS269 in cynomolgus monkey serum (see Example 12), except for the following parameters: (i) for the TA assay, the STD / QC / sample pre-dilution step was 1:100; (ii) for the ADC assay, an MSD high-binding plate was coated with rhPSMA, and the STD / QC / sample pre-dilution step was 1:20; (iii) for the pAF-AS269 assay, dexamethasone-d4 was used as an internal standard, and 100 μL of STD / QC / sample was transferred for the protein precipitation step. The limits of quantitation (LLOQ) for the assays of TA, ADC, and pAF-AS269 in human serum were 62.5 ng / mL, 7.8 ng / mL, and 0.02 ng / mL, respectively. PK parameters were determined using non-compartmental analysis based on the serum concentrations of TA, ADC, and pAF-AS269.

[0310] ARX517 exhibited nearly overlapping TA and ADC PK concentration-time curves at all tested dose levels, indicating strong ADC stability with minimal premature release of the free payload. A long ADC terminal half-life of approximately 6 to 10 days was observed at doses ≥1.4 mg / kg, maximizing drug exposure over a 3-week dosing cycle. Low concentrations of pAF-AS269 (approximately 0.02 ng / mL to 0.2 ng / mL) were observed at all dose levels and appeared slowly in circulation, with Cmax observed approximately 7 days post-administration. This contrasts with other ADCs that typically exhibit free payload Cmax hours to days post-administration.

[0311] ARX517 is the first anti-PSMA ADC tested in a clinical setting, demonstrating remarkable stability and a long terminal half-life. These properties enable continuous drug delivery throughout the dosing cycle, potentially improving efficacy and minimizing toxicity due to premature release of the free payload, thus indicating a clear and favorable therapeutic index.

[0312] The U.S. Food and Drug Administration (FDA) has granted Fast Track designation to the anti-PSMA antibody-drug conjugate (ADC) investigational therapy ARX517 for the treatment of patients with metastatic castration-refractory prostate cancer (mCRPC) who have progressed on androgen receptor pathway inhibitors.

[0313] Example 14: ARX517 solution for intravenous infusion ARX517 ADC formulation development includes ARX517 chemical and physical characterization (e.g., amino acid sequence, charge, isoelectric point (pI), molecular weight, drug-to-antibody ratio (DAR), size, and charge variants), solubility, excipient screening (e.g., excipient properties and concentration, buffer pH, properties, and concentration), and stability studies, as well as consideration of the ability to minimize manufacturing and processing steps between the active pharmaceutical ingredient (ARX517 ADC) and the formulated drug product. The selection of a primary clinical formulation is based on the results of the foregoing assessments, including solubility (protein recovery), chemical, and physical stability criteria using high-performance liquid chromatography (HPLC) and capillary electrophoresis.

[0314] For example, stability tests of ARX517 formulations containing succinate buffer (pH 5.5 or 5.0) versus histidine buffer (pH 5.5, 6.0, and 6.5) after four weeks at 40°C, and analysis of samples by size exclusion chromatography (SEC) to obtain monomer percentage (main peak percentage), high molecular weight (HMW; e.g., containing aggregates) percentage, and low molecular weight (LMW) percentage, showed that the formulations containing succinate buffer had a lower monomer percentage compared to those containing histidine buffer. Notably, the formulations containing succinate buffer had a significantly increased high molecular weight (HMW) percentage (0.91% to 1.69%) compared to those containing histidine buffer (0.52% to 0.55%), indicating a higher tendency for aggregation in the formulations containing succinate buffer. Based on this analysis, the formulation containing succinate buffer at pH 5.5 was the least stable, followed by the formulation containing succinate buffer at pH 5.0. However, the formulation containing histidine buffer at pH 5.5 contained a higher percentage of monomeric substances than the formulation containing succinate buffer at the same pH (Table 11).

[0315] Table 11. Main peak purity and impurities of ARX517 formulation determined by SEC-HPLC after 4 weeks at 40°C. Samples stored at 40°C and analyzed by reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) were measured to have a purity equal to the sum of heavy chain (HC)% and light chain (LC)% under reducing conditions. This also indicated that the histidine formulation exhibited better stability characteristics (e.g., higher percentage of intact matter and lower percentage of other impurities) than the succinate formulation. However, in the histidine formulation, the pH 6.5 histidine buffer formulation showed an increased amount of other impurities (data not shown).

[0316] Imaging capillary isoelectric focusing (icIEF) analysis of the formulation maintained at 40°C for 4 weeks showed an increase in acidic charged substances, with a corresponding decrease in the purity of the main peak. Based on this analysis, the ARX517 formulation contains the following amounts of acidic substances: 52.5% and 48.2% (succinate buffer, pH 5.5 and 5.0, respectively); 48.5% acidic substances (histidine buffer, pH 6.5); and 39.4% and 41.6% (histidine buffer, pH 5.5 and 6.0, respectively).

[0317] The drug-to-antibody ratio (DAR) of formulation samples stored at 40°C was also analyzed by hydrophobic interaction chromatography (HIC); formulations containing succinate buffer at pH 5.0 showed a reduced DAR, while formulations at higher pH were more stable under these conditions (Table 12).

[0318] Table 12. DAR values ​​of ARX517 formulation determined by HIC-HPLC after 4 weeks at 40°C. The final formulation selection was based on the impurity distribution assessed by balancing SEC-HPLC, reducing capillary electrophoresis of sodium dodecyl sulfate (CE-SDS), imaging capillary isoelectric focusing (icIEF), and DAR evaluation via hydrophobic interaction chromatography (HIC)-HPLC. Confirmatory stability studies were conducted in histidine buffers at pH 5.6, 5.9, and 6.2 in Type I borosilicate glass vials with chlorobutyl rubber stoppers and aluminum seals to determine the range and robustness of the selected clinical formulation.

[0319] The clinical formulation for intravenous administration and the ARX517 solution for intravenous injection (hereinafter, ARX517 pharmaceutical product) contain ARX517 ADC, which is prepared at a concentration of 10 mg / mL in a solution of 20 mM histidine buffer (L-histidine, L-histidine hydrochloride), 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80; the final solution pH is 5.9 ± 0.3. The excipients comply with the requirements of applicable pharmacopoeia monographs (Ph.Eur., USP / NF, JP, ChP).

[0320] In accordance with the International Council for Harmonisation of Technical Requirements for Manufacturing (ICH) guidelines, the long-term stability of ARX517 pharmaceutical products manufactured under current Good Manufacturing Practices (cGMP) was evaluated at -20°C ± 5°C, accelerated stability at 5°C ± 3°C, and under stress conditions of 25°C / 60% relative humidity (RH). Stability acceptance criteria included measures of appearance, pH, content (protein concentration), potency (combined with ELISA and cell-based assays), purity (including unconjugated mAb, DAR, CE-SDS, size exclusion chromatography (SEC), and cation exchange chromatography (CEX; see Example 17)), and safety measures (endotoxin, sterility, and particle size).

[0321] Long-term stability. The ARX517 pharmaceutical product exhibited the following characteristics over a 36-month period under long-term stability conditions at -20°C ± 5°C (upright storage), with all tests performed at 0 months and 36 months, and most tests additionally performed at 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and 24 months: Appearance: colorless to pale yellow and clear to milky white liquid, substantially free of visible particles; pH range: 5.6 to 6.2 (specifically, 5.8 to 5.9); Fewer than 20 particles ≥25 μm / vial and fewer than 100 particles ≥10 μm / vial; Protein concentration: 9.0 mg / mL to 11.0 mg / mL (specifically, 10.1 mg / mL to 10.7 mg / mL). Within the range; at all time points, both cell-based ELISA and bound ELISA results were >90% of the reference standard; based on CEX purity, which measures the percentage of main peak, acidic peak, and basic peak, indicating ≥40.0% main peak, ≤40.0% acidic peak, and ≤30.0% basic peak over the study duration (specifically, 51.9% to 53.8% main peak, 31.8% to 34.0% acidic peak, and 13.8% to 14.3% basic peak); based on SEC purity, which measures the percentage of monomers (main peak), high molecular weight (HMW) and low molecular weight (LMW) substances, indicating high purity and minimal aggregation over the study duration (98.0% to 98.3% main peak, 1.7% to 1.9%). The drug contained HMW (high molecular weight vapor) and 0.1% to 0.2% LMW (low molecular weight vapor); endotoxin <0.05 endotoxin units (EU) / mg, and no growth was observed, indicating that the drug product remained sterile; the drug-to-antibody ratio (DAR) measured by hydrophobic interaction chromatography (HIC) was in the range of 1.9 to 2.1 (specifically, 2.1 at all measurement time points, including 0 months and 36 months), and unconjugated mAb (also measured by HIC) was 0.2% at each measurement time point (including 0 months and 36 months); and free drug-related impurities were less than 0.10% (w / w). Based on these and other test results, the ARX517 drug product was found to be stable for at least 36 months under long-term storage conditions of -20°C ± 5°C.

[0322] Additional tests were performed at the 48-month time point: Appearance: clear, colorless liquid with no visible particles; pH: 5.6 to 6.2 (specifically, 5.9); Particles ≥25 μm or ≥10 μm: no more than 6000 per vial (specifically, 31 per vial or 558 per vial, respectively); Protein content: 9.0 mg / mL to 11.0 mg / mL (specifically, 10.3 mg / mL); Purity based on CEX (measured by the percentage of main peak, acidic peak, and basic peak). ≥40.0% main peak, ≤40.0% acidic peak, and ≤30.0% basic peak (specifically, 52.2% main peak, 33.7% acidic substance peak, and 14.1% basic substance peak); SEC-based purity indicates high purity and minimal aggregation (98.2% main peak, 1.7% HMW substance, and 0.1% LMW substance); DAR in the range of 1.9 to 2.1 (specifically, 2.1); unconjugated mAb is 0.2%; and free drug-related impurities are less than 0.10% (w / w) (specifically, below the lower limit of quantitation). Based on these and other test results, the ARX517 drug product was found to be stable for at least 48 months under long-term storage conditions of -20°C ± 5°C.

[0323] Accelerated stability. The ARX517 drug product exhibited the following characteristics over a 6-month period under accelerated stability conditions at 5°C ± 3°C (upright storage), with all tests performed at 0 and 6 months, and most additional tests at 1 and 3 months: Appearance: colorless to pale yellow and clear to milky white liquid, substantially free of visible particles; pH range: 5.6 to 6.2 (specifically, 5.8 to 5.9); Fewer than 5 particles ≥25µm and fewer than 100 particles ≥10µm; Protein concentration: 9.0 mg / mL to 11.0 mg / mL (specifically, 10.1 mg / mL to 10.4 mg / mL); At all time points, the basic... The ELISA results for cells and the combined ELISA results were each >85% of the reference standard; based on the purity of the CEX, which measures the percentage of the main peak, acidic peak, and basic peak, indicating ≥40.0% of the main peak, ≤40.0% of the acidic peak, and ≤30.0% of the basic peak during the study period (specifically, 51.2% to 54.1% of the main peak, 31.8% to 34.5% of the acidic peak, and 14.1% to 14.3% of the basic peak); based on the purity of the SEC, which measures the percentage of monomers (main peak), high molecular weight (HMW) and low molecular weight (LMW) substances, the percentage of the main peak was 97.5% to 98.2% and the basic molecular weight (LMW) was 1.7% to 2.4% during the study period. HMW substance and 0.1% to 0.2% LMW substance; endotoxin < 0.05 EU / mg, and no growth was observed, indicating that the drug product remained sterile; the drug-to-antibody ratio (DAR) as measured by hydrophobic interaction chromatography (HIC) was in the range of 1.9 to 2.1 (specific...

Claims

1. A method for treating cancer, the method comprising: Administer an effective amount of an anti-PSMA antibody-drug conjugate (ADC) to a human subject in need, wherein the anti-PSMA ADC comprises: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain contains a heavy chain variable region of SEQ ID NO: 1, and wherein one non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number; wherein one drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: ； The effective amount of the anti-PSMA ADC is at least about 1.4 mg / kg and at most about 5.0 mg / kg of human subject weight.

2. The method of claim 1, wherein each light chain comprises the light chain variable region of SEQ ID NO:

2.

3. The method according to claim 1 or claim 2, wherein each heavy chain amino acid sequence is SEQ ID NO:8, and the heavy chain amino acid sequence contains pAF at Kabat position 114.

4. The method according to any one of claims 1 to 3, wherein each light chain amino acid sequence is SEQ ID NO:

9.

5. The method according to any one of claims 1 to 4, wherein the effective amount of the anti-PSMA ADC is at least about 1.4 mg / kg and at most about 3.4 mg / kg of the human subject's body weight.

6. The method according to any one of claims 1 to 4, wherein the effective amount of the anti-PSMA ADC is about 1.4 mg / kg, about 1.7 mg / kg, about 2 mg / kg, about 2.4 mg / kg, about 2.9 mg / kg, about 3.2 mg / kg, about 3.4 mg / kg, about 3.5 mg / kg, about 4.3 mg / kg, about 4.5 mg / kg, about 4.7 mg / kg, or about 5 mg / kg of the human subject's body weight.

7. The method according to any one of claims 1 to 6, wherein the effective amount of the anti-PSMA ADC is greater than 2.0 mg / kg of the human subject's body weight.

8. The method according to any one of claims 1 to 7, wherein the anti-PSMA ADC is applied once every three weeks or once every four weeks.

9. The method according to any one of claims 1 to 8, wherein the human subject has prostate cancer expressing PSMA or non-prostate cancer expressing PSMA.

10. The method according to any one of claims 1 to 9, wherein the human subject has prostate cancer.

11. The method of claim 10, wherein the prostate cancer is metastatic castration-refractory prostate cancer (mCRPC).

12. The method according to any one of claims 9 to 11, wherein the prostate cancer progresses after prior taxane therapy.

13. The method according to any one of claims 1 to 12, wherein the cancer is hormone-resistant prostate cancer.

14. The method according to any one of claims 1 to 13, wherein the method delays or inhibits the progression of the cancer in the human subject.

15. The method according to any one of claims 1 to 14, wherein the method increases the survival of the human subject compared to the median survival of subjects who have not previously been treated with the same or different anti-PSMA ADCs.

16. The method according to any one of claims 1 to 15, wherein the method reduces the circulating level of CTCs in the human subject compared to the baseline level of circulating tumor cells (CTCs) in the human subject.

17. The method according to any one of claims 1 to 16, wherein the method reduces or stabilizes serum PSA levels in the human subject compared to baseline levels of prostate-specific antigen (PSA) in the human subject.

18. The method of claim 17, wherein the reduction in serum PSA levels in the human subject is at least about 30% lower than the baseline PSA levels in the human subject.

19. The method of claim 17, wherein the reduction in serum PSA levels in the human subject is at least about 50% of the reduction in serum PSA levels in the human subject compared to baseline PSA levels.

20. The method according to any one of claims 1 to 19, wherein the administration is intravenous.

21. The method according to any one of claims 1 to 20, wherein the cancer is resistant or refractory to prior standard therapies.

22. The method according to any one of claims 1 to 21, wherein the human subject has previously been treated with abiraterone, dalolutamide, apalutamide, or enzalutamide.

23. The method according to any one of claims 1 to 22, further comprising administering an effective amount of an additional therapeutic agent.

24. The method of claim 23, wherein the additional therapeutic agent is a chemotherapeutic agent, a hormonal agent, an antitumor agent, an immunostimulant, an immunomodulator, an immunotherapeutic agent, or a combination thereof.

25. The method of claim 24, wherein the hormone agent is enzalutamide.

26. The method of any one of claims 1 to 25, wherein the method increases the survival of the human subject, wherein the survival of the human subject is increased compared to the median survival of subjects with taxane-resistant cancer expressing anti-PSMA who have not previously been treated with the same or different anti-PSMA ADCs.

27. The method according to any one of claims 1 to 26, wherein after administering the effective amount of anti-PSMA ADC to the human subject, the method provides a serum terminal half-life of at least about 5 days for the anti-PSMA ADC.

28. The method according to any one of claims 1 to 27, wherein after administering the effective amount of the anti-PSMA ADC to the human subject, the treatment method provides a time (Tmax) of at least about 5 days to reach the maximum concentration of the free payload in serum, wherein the free payload has the following structure: ； Or its salt.

29. The method according to any one of claims 1 to 28, wherein after administering the effective amount of the anti-PSMA ADC to the human subject, the treatment method provides a maximum serum concentration (Cmax) of up to about 1 ng / mL of free payload, wherein the free payload has the following structure: ； Or its salt.

30. The method of claim 29, wherein after administering the effective amount of anti-PSMAADC to the human subject, the serum effective load Cmax is at most about 0.5 ng / mL, at most about 0.4 ng / mL, at most about 0.3 ng / mL, or at most about 0.2 ng / mL.

31. The method according to any one of claims 1 to 30, wherein after administering the effective amount of anti-PSMA ADC to the human subject, the treatment method provides a serum maximum concentration (Cmax) of at least about 20 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL of anti-PSMA ADC.

32. The method according to any one of claims 1 to 31, wherein after administering the effective amount of the anti-PSMA ADC to the human subject, the treatment provides at least about 50% reduction in circulating tumor DNA (ctDNA).

33. The method according to any one of claims 1 to 32, wherein the anti-PSMA ADC is ARX517.

34. A pharmaceutical composition comprising an effective amount of an anti-PSMA antibody-drug conjugate (ADC), wherein the anti-PSMA ADC comprises: A humanized anti-PSMA monoclonal antibody comprising two heavy chains and two light chains, wherein each heavy chain contains a heavy chain variable region of SEQ ID NO:1, wherein a non-natural amino acid, p-acetyl-L-phenylalanine (pAF), is incorporated into each heavy chain at position 114 according to the Kabat number, and each light chain contains a light chain variable region of SEQ ID NO:2; wherein a drug-linker is conjugated to each pAF via an oxime bond, wherein each drug-linker is amberstatin269 (AS269) having the following structure: ， The concentration of the anti-PSMA ADC is in the range of about 5 mg / mL to about 25 mg / mL. And one or more pharmaceutically acceptable components selected from the group consisting of sucrose, histidine buffer and polysorbate 80 and combinations thereof, wherein the pH of said composition is in the range of about 5.5 to about 6.

5.

35. The pharmaceutical composition according to claim 34, wherein each heavy chain amino acid sequence is SEQ ID NO: 8, the heavy chain amino acid sequence contains pAF at Kabat position 114, and / or each light chain amino acid sequence is SEQ ID NO:

9.

36. The pharmaceutical composition according to claim 34 or claim 35, wherein the concentration of anti-PSMA ADC is about 10 mg / mL.

37. The pharmaceutical composition according to any one of claims 34 to 36, wherein the sucrose concentration is in the range of about 5% (w / v) to about 15% (w / v); the histidine buffer concentration is in the range of about 15 mM to about 25 mM; and the polysorbate 80 concentration is in the range of about 0.001% (w / v) to about 0.02% (w / v).

38. The pharmaceutical composition according to any one of claims 34 to 37, wherein the composition comprises essentially the following: an anti-PSMA ADC at a concentration of about 10 mg / mL; sucrose at a concentration of about 9% (w / v); histidine buffer at a concentration of about 20 mM; and polysorbate 80 at a concentration of about 0.01% (w / v); wherein the pH of the composition is about 5.9 ± 0.

3.

39. The pharmaceutical composition according to any one of claims 34 to 38, wherein the composition is a liquid formulation.

40. The pharmaceutical composition according to any one of claims 34 to 39, wherein the anti-PSMA ADC comprises a charged variant, wherein the charged variant comprises an anti-PSMA ADC main substance, an anti-PSMA ADC acidic substance, and an anti-PSMA ADC basic substance.

41. The pharmaceutical composition of claim 40, wherein the main anti-PSMA ADC substance has an isoelectric point (pI) of about 8.3, the acidic anti-PSMA ADC substance has a pI of about 8.1, and the basic anti-PSMA ADC substance has a pI of about 8.

4.

42. The pharmaceutical composition according to claim 40 or 41, wherein the anti-PSMA ADC major substance is present in an amount of about 40% to about 70%, the anti-PSMA ADC acidic substance is present in an amount of about 20% to about 40%, and the anti-PSMA ADC basic substance is present in an amount of about 5% to about 30%; wherein the sum of the percentages of each of the major substance, the acidic substance, and the basic substance is 100%.

43. The pharmaceutical composition according to any one of claims 34 to 42, wherein the drug-to-antibody ratio (DAR) is in the range of about 1.5 to about 2.

5.

44. The pharmaceutical composition of claim 43, wherein the drug-to-antibody ratio (DAR) is in the range of about 1.9 to about 2.

1.

45. The pharmaceutical composition according to any one of claims 34 to 44, wherein the anti-PSMA ADC is ARX517.

46. ​​The pharmaceutical composition according to any one of claims 34 to 45, wherein the pharmaceutical composition is used in the method according to any one of claims 1 to 33.

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