CN116710071A

CN116710071A - Subcutaneous administration of PD1/PD-L1 antibodies

Info

Publication number: CN116710071A
Application number: CN202180088287.6A
Authority: CN
Inventors: M·黄; M·霍斯萨维; T·A·哈比; S·A·哈特; R·V·曼特利; H·E·维齐纳; A·罗伊; B·P·默西; U·A·阿拉斯; K·桑加维; X·赵; A·贝洛
Original assignee: Olozme Co; Bristol Myers Squibb Co
Current assignee: Olozme Co; Bristol Myers Squibb Co
Priority date: 2020-12-28
Filing date: 2021-12-27
Publication date: 2023-09-05

Abstract

The present disclosure provides pharmaceutical compositions comprising an anti-PD-1 antibody or an anti-PD-L1 antibody formulated for subcutaneous delivery, wherein the pharmaceutical composition further comprises an endoglycosidase hydrolase, such as a hyaluronidase.

Description

Subcutaneous administration of PD1/PD-L1 antibodies

Cross Reference to Related Applications

The application requires U.S. provisional application No. US 63/131,240 filed on 12/28 of 2020; U.S. provisional application No. US 63/150,420 filed on day 17 of 2 of 2021; priority and equity of U.S. provisional application No. US 63/184,082 filed on 5/4 of 2021; each of which is hereby incorporated by reference in its entirety.

Reference to sequence Listing submitted electronically via EFS-WEB

The contents of the electronically submitted sequence listing (name 3338_225PC03_Seqling_ST25. Txt; size 1,011,316 bytes; date of creation 2021, 12, 27) are incorporated herein by reference in their entirety.

Technical Field

The present disclosure provides a method for treating a subject having a tumor with a checkpoint inhibitor (e.g., immunotherapy).

Background

Human cancers have many genetic and epigenetic changes, producing new antigens that are potentially recognizable by the immune system (Sjoblom et al, science (2006) 314 (5797): 268-274). The adaptive immune system, consisting of T lymphocytes and B lymphocytes, has a strong anticancer potential, a broad capacity and precise specificity to respond to a wide variety of tumor antigens. Furthermore, the immune system exhibits considerable plasticity and memory components. Successful exploitation of all of these attributes of the adaptive immune system would make immunotherapy unique among all cancer treatments.

Until recently, cancer immunotherapy has focused a great deal of effort on methods of enhancing anti-tumor immune responses by adoptively transferring activated effector cells, immunizing against related antigens, or providing nonspecific immunostimulants (e.g., cytokines). However, in the last decade, a great deal of effort to develop specific immune checkpoint pathway inhibitors has begun to provide new immunotherapeutic approaches for the treatment of cancer, including the development of antibodies (such as nivolumab and pembrolizumab) (previously lambrolizumab; USAN committee statement, 2013)) that specifically bind to the programmed death protein-1 (PD-1) receptor and block the inhibitory PD-1/PD-1 ligand pathway (Topalian et al 2012a, b; topalian et al 2014; hamid et al 2013; hamid and Carvajal 2013; mcdermott and Atkins, 2013).

Current methods of delivering anti-PD-1 and/or anti-PD-L1 antibodies use periodic intravenous administration, administered by a clinician, typically at a clinic or hospital. The inconvenience and invasiveness of the treatment may negatively impact the patient's experience. Subcutaneous delivery (e.g., by using an auto-injector or wearable pump) can significantly improve patient compliance. However, there remains a need in the art for formulations comprising anti-PD-1 or anti-PD-L1 antibodies suitable for subcutaneous delivery to a patient.

Disclosure of Invention

Certain aspects of the present disclosure relate to a method of treating a subject in need thereof, the method comprising subcutaneously administering to the subject a dose of a pharmaceutical composition comprising (i) an antibody that specifically binds PD-1 or PD-L1 and inhibits the interaction of PD-1 with PD-L1 (an "anti-PD-1 antibody" or an "anti-PD-L1 antibody", respectively) and (ii) an endoglycosidase hydrolase; wherein the dose comprises one or more subcutaneous unit doses; and wherein the dose comprises at least about 300mg to at least about 2400mg of the anti-PD-1 antibody or the anti-PD-L1 antibody.

In some aspects, the dose comprises two or more subcutaneous unit doses, wherein the two or more subcutaneous unit doses are administered in parallel or subsequently. In some aspects, the two or more subcutaneous unit doses are administered sequentially, wherein each of the two or more subcutaneous unit doses is administered within an interval of about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about nine hours, about twelve hours, about eighteen hours, or about twenty four hours between the subcutaneous unit doses. In some aspects, the dose is administered about every, two, three, four, six, eight weeks.

In some aspects, the antibody comprises an anti-PD-1 antibody. In some aspects, the antibody is administered at a dose of about 250mg to about 600mg per week. In some aspects, the antibody is administered at a dose of about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, or about 600mg per week. In some aspects, the antibody is administered at a dose of about 300mg about weekly. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 300mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 300mg in a total administration volume of about 2 mL.

In some aspects, the dosage of the antibody comprises (i) two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 150mg of the antibody; or (ii) three subcutaneous unit doses, wherein each of the three subcutaneous unit doses comprises about 100mg of the antibody. In some aspects, (i) the two subcutaneous unit doses are administered to the same body location of the subject, or (ii) at least two of the three subcutaneous unit doses are administered to the same body location of the subject.

In some aspects, the antibody is administered at a dose of about 300mg to about 900mg every two weeks. In some aspects, the antibody is administered at a dose of about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 730mg, about 740mg, about 750mg, about 760mg, about 770mg, about 780mg, about 790mg, about 800mg, about 810mg, about 820mg, about 830mg, about 840mg, about 850mg, about 860mg, about 870mg, about 880mg, about 890mg, or about 900mg every two weeks. In some aspects, the antibody is administered at a dose of about 600mg every two weeks. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose.

In some aspects, the dosage of the antibody comprises two, three, or at least four subcutaneous unit doses. In some aspects, the dosage of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 300mg of the antibody. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least two of the subcutaneous unit doses are administered to the same body location of the subject.

In some aspects, the antibody is administered at a dose of about 900mg to about 1500mg every four weeks. In some aspects, the antibody is administered at a dose of about 900, about 950, about 1000mg, about 1010mg, about 1020mg, about 1030mg, about 1040mg, about 1050mg, about 1060mg, about 1070mg, about 1080mg, about 1090mg, about 1100mg, about 1110mg, about 1120mg, about 1130mg, about 1140mg, about 1150mg, about 1160mg, about 1170mg, about 1180mg, about 1190mg, about 1200mg, about 1210mg, about 1220mg, about 1230mg, about 1240mg, about 1250mg, about 1260mg, about 1270mg, about 1280mg, about 1290mg, about 1300mg, about 1310mg, about 1320mg, about 1330mg, about 1340mg, about 1350mg, about 1360mg, about 1370mg, about 1380mg, about 1390mg, about 1400mg, about 1410mg, about 1420mg, about 1430mg, about 1440mg, about 1450mg, about 0mg, about 148 mg, or about 1500mg. In some aspects, the antibody is administered at a dose of about 1200mg every four weeks. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose.

In some aspects, the dosage of the antibody comprises two, three, four, six, or at least eight subcutaneous unit doses. In some aspects, the dosage of the antibody comprises four subcutaneous unit doses, wherein each of the four subcutaneous unit doses comprises about 300mg of the antibody. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of 2 mL. In some aspects, at least two of the subcutaneous unit doses are administered to the same body location of the subject. In some aspects, the two, three, four, six, or at least eight subcutaneous unit doses are administered on the same day.

In some aspects, the antibody comprises an anti-PD-L1 antibody. In some aspects, the antibody is administered at a dose of about 900mg to about 1800mg of the antibody every two weeks. In some aspects, the antibody is administered at a dose of about 900, about 950, about 1000, about 1010, about 1020, about 1030, about 1040, about 1050, about 1060, about 1070, about 1080, about 1090, about 1100, about 1110, about 1120, about 1130, about 1140, about 1150, about 1160, about 1170, about 1180, about 1190, about 1200, about 1210, about 1220, about 1230, about 1240, about 1250, about 1260, about 1270, about 1280, about 1290, about 1300, about 1310, about 1320, about 1330, about 1340, about 1350, about 1360, about 1370, about 1380, about 1390, about 1400, about 1410, about 1420, about 1430, about 1440, about 1450, about 0, about 148, about 1500, about 148 mg, about 1500, about six weeks. In some aspects, the dosage of the antibody is about 1200mg every two weeks. In some aspects, the dose comprises a single subcutaneous unit dose of greater than about 1200mg in a total administered volume of about 5 mL.

In some aspects, the dose comprises two, three, four, six, or at least eight subcutaneous unit doses. In some aspects, the dosage of the antibody comprises four subcutaneous unit doses, wherein each of the four subcutaneous unit doses comprises about 300mg of the antibody. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least two of the subcutaneous unit doses are administered to the same body location of the subject. In some aspects, the two, three, four, six, or at least eight subcutaneous unit doses are administered on the same day.

In some aspects, the anti-PD-1 antibody or anti-PD-L1 antibody and the endoglycosidase hydrolase are administered together. In some aspects, the anti-PD-1 antibody or anti-PD-L1 antibody and the endoglycosidase hydrolase are administered concurrently. In some aspects, the anti-PD-1 antibody or anti-PD-L1 antibody and the endoglycosidase hydrolase are administered sequentially.

In some aspects, the pharmaceutical composition is administered at a dose of at least about 1,000 units to at least about 30,000 units of the endoglycosidase hydrolase. In some aspects, the pharmaceutical composition is administered in at least about 1000 units, at least about 1500 units, at least about 2000 units, at least about 2500 units, at least about 3000 units, at least about 3500 units, at least about 4000 units, at least about 4500 units, at least about 5000 units, at least about 6000 units, at least about 7000 units, at least about 8000 units, at least about 9000 units, at least about 10,000 units, at least about 11,000 units, at least about 12,000 units, at least about 13,000 units, at least about 14,000 units, at least about 15,000 units, at least about 16,000 units, at least about 17,000 units, at least about 18,000 units, at least about 19,000 units, at least about 20,000 units, at least about 21,000 units, at least about 22,000 units, at least about 23,000 units, at least about 24,000 units, at least about 25,000 units, at least about 26,000 units, at least about 27,000 units, at least about 28,000 units, at least about 29,000 units, or at least about 30,000 units of the endoglycosidase. In some aspects, the pharmaceutical composition is administered at a dose of about 4000 units of the endoglycosidase hydrolase.

In some aspects, the pharmaceutical composition is administered at the following doses: (i) About 300mg of the antibody and (ii) about 4000 units of the endoglycosidase hydrolase, about once a week. In some aspects, the pharmaceutical composition is administered at the following doses: (i) About 600mg of the antibody and (ii) about 8000 units of the endoglycosidase hydrolase, about once every two weeks. In some aspects, the pharmaceutical composition is administered at the following doses: (i) About 1200mg of the antibody and (ii) about 20,000 units of the endoglycosidase hydrolase, about once every four weeks. In some aspects, the pharmaceutical composition is administered at the following doses: (i) About 1200mg of the antibody and (ii) about 8000 units of the endoglycosidase hydrolase, about once every two weeks. In some aspects, the pharmaceutical composition is administered at the following doses: (i) About 1800mg of said antibody and (ii) about 12,000 units of said endoglycosidase hydrolase, about once every three weeks.

In some aspects, the endoglycosidase hydrolase cleaves hyaluronic acid at a hexosamine β (1-4) or (1-3) junction. In some aspects, the endoglycosidase hydrolase comprises the catalytic domain of hyaluronidase PH-20 (HuPH 20), HYAL1, HYAL2, HYAL3, HYAL4, or HYALPS 1. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to amino acids 36-490 of SEQ ID No. 1. In some aspects, the endoglycosidase hydrolase comprises a hyaluronidase. In some aspects, the endoglycosidase hydrolase comprises a hyaluronidase selected from the group consisting of HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, any variant thereof, and any isoform. In some aspects, the endoglycosidase hydrolase comprises rHuPH20 or a fragment thereof.

In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the alpha helical region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the linker region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase in which one or more N-terminal and/or C-terminal amino acids are deleted relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises: i. one or more amino acid substitutions in the alpha helical region, the linker region, or both the alpha helical region and the linker region relative to wild-type rHuPH 20; deletion of one or more N-terminal amino acids, one or more C-terminal amino acids, or one or more N-terminal amino acids and one or more C-terminal amino acids relative to wild-type rHuPH 20; or both (i) and (ii).

In some aspects, the anti-PD-1 antibody comprises an antibody selected from the group consisting of: nivolumab, pembrolizumab, PDR001, MEDI-0680, cimetidine Li Shan, terlipressin Li Shan, tirelimumab, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, BCD-100, IBI308, and any combination thereof. In some aspects, the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1. In some aspects, the anti-PD-1 antibody comprises nivolumab. In some aspects, the anti-PD-1 antibody comprises pembrolizumab.

In some aspects, the anti-PD-L1 antibody comprises an antibody selected from the group consisting of: BMS-936559, ab, duvaluzumab, ab, STI-1014, CX-072, KN035, LY3300054, BGB-A333, CK-301, and any combination thereof.

In some aspects, the subject has cancer. In some aspects of the present invention, the cancer is selected from squamous cell carcinoma (squamous cell carcinoma), small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (renal cancer), clear cell carcinoma, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer (endometrial cancer), renal cancer (kidney cancer), renal Cell Carcinoma (RCC), prostate cancer, renal cancer, and renal cancer hormone refractory prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer (cervical cancer), gastric cancer (cancer), bladder cancer, hepatoma, breast cancer, colon cancer, head and neck cancer, gastric cancer (cancer), germ cell tumors, pediatric sarcoma, natural killer cells of the sinuses melanoma, bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer (carcinoma of the endometrium), cervical cancer (carcinoma of the cervix), vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, rectal cancer, childhood solid tumor, ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma (squamous cell cancer), environmentally-induced cancers including asbestos-induced cancers, virus-related cancers, or cancers of viral origin (e.g., human papillomavirus (HPV-associated or derived tumor)), and any combination thereof.

In some aspects, the pharmaceutical composition is administered using an automatic injector. In some aspects, the pharmaceutical composition is administered using a wearable pump.

In some aspects, the pharmaceutical composition is administered to the subject by subcutaneous infusion for less than about 10 minutes. In some aspects, the pharmaceutical composition is administered to the subject by subcutaneous infusion for less than about 5 minutes.

In some aspects, the pharmaceutical composition further comprises at least two antioxidants. In some aspects, the at least two antioxidants are selected from methionine, tryptophan, histidine, cysteine, ascorbic acid, glycine, DTPA, and EDTA. In some aspects, the at least two antioxidants comprise (i) methionine and EDTA, or (ii) methionine and DTPA. In some aspects, the at least two antioxidants comprise at least about 1 to about 20mM methionine. In some aspects, the at least two antioxidants comprise at least about 1mM, at least about 1.5mM, at least about 2mM, at least about 2.5mM, at least about 3mM, at least about 3.5mM, at least about 4mM, at least about 4.5mM, at least about 5mM, at least about 5.5mM, at least about 6mM, at least about 6.5mM, at least about 7mM, at least about 7.5mM, at least about 8mM, at least about 8.5mM, at least about 9mM, at least about 9.5mM, at least about 10mM, at least about 11mM, at least about 12mM, at least about 13mM, at least about 14mM, at least about 15mM, at least about 16mM, at least about 17mM, at least about 18mM, at least about 19mM, or at least about 20mM methionine. In some aspects, the at least two antioxidants comprise about 5mM methionine.

In some aspects, the at least two antioxidants comprise at least about 10 μm to about 200 μm DTPA. In some aspects, the at least two antioxidants comprise at least about 10 μΜ, at least about 15 μΜ, at least about 20 μΜ, at least about 25 μΜ, at least about 30 μΜ, at least about 35 μΜ, at least about 40 μΜ, at least about 45 μΜ, at least about 50 μΜ, at least about 55 μΜ, at least about 60 μΜ, at least about 65 μΜ, at least about 70 μΜ, at least about 75 μΜ, at least about 80 μΜ, at least about 85 μΜ, at least about 90 μΜ, at least about 95 μΜ, at least about 100 μΜ, at least about 110 μΜ, at least about 120 μΜ, at least about 130 μΜ, at least about 140 μΜ, at least about 150 μΜ, at least about 160 μΜ, at least about 170 μΜ, at least about 180 μΜ, at least about 190 μΜ, or at least about 200 μΜ DTPA. In some aspects, the at least two antioxidants comprise about 50 μm DTPA.

In some aspects, the pharmaceutical composition further comprises a tonicity modifier and/or stabilizer. In some aspects, the tonicity modifier and/or stabilizer includes a sugar, amino acid, polyol, salt, or combination thereof. In some aspects, the tonicity modifier and/or stabilizer is selected from the group consisting of sucrose, sorbitol, trehalose, mannitol, glycerol, glycine, leucine, isoleucine, sodium chloride, proline, arginine, histidine, and any combination thereof. In some aspects, the tonicity modifier includes sucrose. In some aspects, the pharmaceutical composition comprises at least about 10mM to at least about 500mM sucrose. In some aspects, the pharmaceutical composition comprises at least about 10mM, at least about 20mM, at least about 30mM, at least about 40mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, at least about 100mM, at least about 110mM, at least about 120mM, at least about 130mM, at least about 140mM, at least about 150mM, at least about 160mM, at least about 170mM, at least about 180mM, at least about 190mM, at least about 200mM, at least about 210mM, at least about 220mM, at least about 230mM, at least about 240mM, at least about 250mM, at least about 260mM, at least about 270mM, at least about 280mM, at least about 290mM, at least about 300mM, at least about 310mM, at least about 320mM, at least about 330mM, at least about 340mM, at least about 350mM, at least about 360mM, at least about 370mM, at least about 380mM, at least about 400mM, at least about 410mM, at least about 420mM, at least about 430mM, at least about 450mM, at least about 460mM, at least about 470mM, at least about 500mM, or at least about 500 mM. In some aspects, the pharmaceutical composition comprises about 250mM sucrose.

In some aspects, the pharmaceutical composition further comprises a buffer. In some aspects, the buffer is selected from histidine, succinate, tromethamine, sodium phosphate, sodium acetate, and sodium citrate. In some aspects, the buffer comprises histidine. In some aspects, the pharmaceutical composition comprises at least about 5mM to at least about 100mM histidine. In some aspects, the pharmaceutical composition comprises at least about 5mM, at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, or at least about 100mM histidine. In some aspects, the pharmaceutical composition comprises about 20mM histidine.

In some aspects, the pharmaceutical composition further comprises a surfactant. In some aspects, the surfactant is selected from polysorbate 20, polysorbate 80, and poloxamer 188. In some aspects, the surfactant comprises polysorbate 80. In some aspects, the pharmaceutical composition comprises at least about 0.01% w/v to at least about 0.1% w/v polysorbate 80. In some aspects, the pharmaceutical composition comprises at least about 0.01% w/v, at least about 0.02% w/v, at least about 0.03% w/v, at least about 0.04% w/v, at least about 0.05% w/v, at least about 0.06% w/v, at least about 0.07% w/v, at least about 0.08% w/v, at least about 0.09% w/v, or at least about 0.1% w/v polysorbate 80. In some aspects, the pharmaceutical composition comprises about 0.05% w/v polysorbate 80.

In some aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 0.0182mg/mL rHuPH20.

In some aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 0.0182mg/mL rHuPH20.

In some aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the pharmaceutical composition comprises a pH of about 5.2 to about 6.8. In some aspects, the pharmaceutical composition comprises a pH of 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 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, or about 6.8. In some aspects, the pharmaceutical composition comprises a pH of about 6.0.

Certain aspects of the present disclosure relate to a pharmaceutical composition for use in any of the methods disclosed herein. Certain aspects of the present disclosure relate to a pharmaceutical composition comprising (i) an antibody that specifically binds PD-1 ("anti-PD-1 antibody"), (ii) an endoglycosidase hydrolase, and (iii) at least two antioxidants.

In some aspects, the pharmaceutical composition comprises at least about 50U/mL to at least about 5000U/mL of the endoglycosidase hydrolase. In some aspects, the pharmaceutical composition comprises the glycosidase hydrolase of at least about 50U/mL, at least about 100U/mL, at least about 150U/mL, at least about 200U/mL, at least about 250U/mL, at least about 300U/mL, at least about 350U/mL, at least about 400U/mL, at least about 450U/mL, at least about 500U/mL, at least about 750U/mL, at least about 1000U/mL, at least about 1500U/mL, at least about 2000U/mL, at least about 2500U/mL, at least about 3000U/mL, at least about 3500U/mL, at least about 4000U/mL, at least about 4500U/mL, at least about 5000U/mL, at least about 5500U/mL, at least about 6000U/mL, at least about 6500U/mL, at least about 7000U/mL, at least about 7500U/mL, at least about 8000U/mL, at least about 8500U/mL, at least about 9000U/mL, at least about 9500U/mL, at least about 10,000U/mL. In some aspects, the pharmaceutical composition comprises at least about 500U/mL of the endoglycosidase hydrolase. In some aspects, the pharmaceutical composition comprises at least about 1000U/mL of the endoglycosidase hydrolase. In some aspects, the pharmaceutical composition comprises at least about 2000U/mL of the endoglycosidase hydrolase.

In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the alpha helical region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the linker region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase in which one or more N-terminal and/or C-terminal amino acids are deleted relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises: (i) One or more amino acid substitutions in the alpha helical region, the linker region, or both the alpha helical region and the linker region relative to wild-type rHuPH 20; (ii) Deletion of one or more N-terminal amino acids, one or more C-terminal amino acids, or one or more N-terminal amino acids and one or more C-terminal amino acids relative to wild-type rHuPH 20; or (iii) both (i) and (ii).

In some aspects, the at least two antioxidants are selected from methionine, tryptophan, histidine, cysteine, ascorbic acid, glycine, DTPA, and EDTA. In some aspects, the at least two antioxidants comprise (i) methionine and EDTA, or (ii) methionine and DTPA. In some aspects, the at least two antioxidants comprise at least about 1 to about 20mM methionine. In some aspects, the at least two antioxidants comprise at least about 1mM, at least about 1.5mM, at least about 2mM, at least about 2.5mM, at least about 3mM, at least about 3.5mM, at least about 4mM, at least about 4.5mM, at least about 5mM, at least about 5.5mM, at least about 6mM, at least about 6.5mM, at least about 7mM, at least about 7.5mM, at least about 8mM, at least about 8.5mM, at least about 9mM, at least about 9.5mM, at least about 10mM, at least about 11mM, at least about 12mM, at least about 13mM, at least about 14mM, at least about 15mM, at least about 16mM, at least about 17mM, at least about 18mM, at least about 19mM, or at least about 20mM methionine. In some aspects, the at least two antioxidants comprise about 5mM methionine.

In some aspects, the pharmaceutical composition comprises at least about 20mg/mL to at least about 200mg/mL of the anti-PD-1 antibody. In some aspects, the pharmaceutical composition comprises at least about 135mg/mL to at least about 180mg/mL of the anti-PD-1 antibody. In some aspects, the pharmaceutical composition comprises at least about 108mg/mL to at least about 132mg/mL of the anti-PD-1 antibody. In some aspects, the pharmaceutical composition comprises at least about 20mg/mL, at least about 30mg/mL, at least about 40mg/mL, at least about 50mg/mL, at least about 60mg/mL, at least about 70mg/mL, at least about 80mg/mL, at least about 90mg/mL, at least about 100mg/mL, at least about 110mg/mL, at least about 120mg/mL, at least about 130mg/mL, at least about 140mg/mL, at least about 150mg/mL, at least about 160mg/mL, at least about 170mg/mL, at least about 180mg/mL, at least about 190mg/mL, or at least about 200mg/mL of the anti-PD-1 antibody. In some aspects, the pharmaceutical composition comprises about 120mg/mL of the anti-PD-1 antibody. In some aspects, the pharmaceutical composition comprises about 150mg/mL of the anti-PD-1 antibody.

In some aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

Certain aspects of the present disclosure relate to a unit dose comprising any of the pharmaceutical compositions disclosed herein.

Certain aspects of the present disclosure relate to a unit dose comprising: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

Certain aspects of the present disclosure relate to a unit dose comprising: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the pharmaceutical composition further comprises a second therapeutic agent. In some aspects, the second therapeutic agent is an antibody. In some aspects, the second therapeutic agent is a checkpoint inhibitor. In some aspects, the checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM 3 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, an anti-NKG 2a antibody, an anti-OX 40 antibody, an anti-ICOS antibody, an anti-MICA antibody, an anti-CD 137 antibody, an anti-KIR antibody, an anti-tgfβ antibody, an anti-IL-10 antibody, an anti-IL-8 antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-CXCR 4 antibody, an anti-mesothelin antibody, an anti-CD 27 antibody, an anti-GITR, or any combination thereof. In some aspects, the pharmaceutical composition further comprises a third therapeutic agent. In some aspects, the second therapeutic agent, the third therapeutic agent, or both comprise IL-2 (e.g., bei Peia interleukin (bempeg aldeslicekin)) or IL12-Fc (e.g., BMS-986415).

Certain aspects of the present disclosure relate to a vial comprising any of the pharmaceutical compositions disclosed herein or any of the unit doses disclosed herein.

In some aspects, the vial is an auto-injector. Certain aspects of the present disclosure relate to an auto-injector comprising any of the pharmaceutical compositions disclosed herein or any of the unit doses disclosed herein.

In some aspects, the vial is a wearable pump. Certain aspects of the present disclosure relate to a wearable pump comprising any of the pharmaceutical compositions disclosed herein or any of the unit doses disclosed herein.

Drawings

Figure 1 presents a graphical representation of data relating to osmotic pressure and viscosity of a nivolumab Subcutaneous (SC) injection formulation as a function of sucrose concentration in the formulation according to example 1. The X-axis represents sucrose concentration (mM) and the Y-axis represents formulation osmolality (mOsm/kg). Filled circles and solid lines represent osmotic pressure values, and filled X and dashed lines represent viscosity values.

Figure 2 presents a graphical representation of data relating to the effect of 75mM added arginine on the viscosity of a Subcutaneous (SC) injection formulation of nivolumab according to example 1. The X-axis represents protein concentration (mg/mL) and the Y-axis represents viscosity (cP) at 20 ℃. Solid boxes represent samples containing added arginine, and filled diamonds represent samples without arginine.

Fig. 3 is a schematic of a study involving evaluation of safety and efficacy of various doses of subcutaneously administered anti-PD-1 antibodies (e.g., nivolumab) alone or in combination with hyaluronidase (e.g., rHuPH 20).

FIG. 4 is a line graph illustration of predictive examination of a combined SC/IV PPK model for administration of nivolumab. Individual dots represent observation data. The lines represent the 5 th, 50 th and 95 th percentiles of the observed data, respectively. The shaded area represents the 5 th (lowest trend line), 50 th (middle trend line) and 95 th (highest trend line) percentiles for the predicted data, based on the simulated 90% ci. Conc = concentration; nivo = nivolumab; pred-Corr = prediction corrected.

Fig. 5A-5C are box plots illustrating geometric mean ratios (SC/IV) of Cavgd28 (fig. 5A), cmind28 (fig. 5B) and Cmax1 (fig. 5C) exposures predicted by tumor type. CRC = colorectal cancer; HCC = hepatocellular carcinoma; mel = melanoma; NSCLC = non-small cell lung cancer; and RCC = renal cell carcinoma.

Fig. 6 is a schematic of a study involving evaluation of safety and efficacy of 1200mg of nivolumab in combination with hyaluronidase (e.g., rHuPH 20) administered subcutaneously once every 4 weeks as compared to 3mg/kg of nivolumab administered IV once every 2 weeks.

FIG. 7 is a box plot illustrating the distribution of nivolumab Cmnd 28 in dose and body weight at 3mg/kg of nivolumab administered IV once every 2 weeks, 10mg/kg of nivolumab administered IV once every 2 weeks, and 1200mg of nivolumab administered subcutaneously once every 4 weeks.

FIGS. 8A-8C are box plots illustrating C observed after subcutaneous delivery of nivolumab at 720mg, 960mg or 1200mg with rHuPH20 _avg (FIG. 8A), C _tau (FIG. 8B) and C _max (FIG. 8C) distribution as observed by body weight. The dashed line shows the geometric mean C of nivolumab 3mg/kg IV Q2W (history) _avg (FIG. 8A) and C _tau (FIG. 8B) geometric mean C of nivolumab 10mg/kg IV Q2W (history) _max (FIG. 8C).

Fig. 9A-9B show tumor-infiltrating lymphocyte CD8 expression (fig. 9A) and PD-L1 tumor expression (fig. 9B) 14 days after the first subcutaneous dose of nivolumab and rHuPH20 (parts A, B and D) for subjects with non-small cell lung cancer (NSCLC), renal Cell Carcinoma (RCC), melanoma (Mel), hepatocellular carcinoma (HCC) and microsatellite instability/mismatch repair deficient colorectal cancer (MSI-H/dMMR CRC).

Fig. 10 is a graphical representation of the effect of headspace nitrogen and air on% HMW species for Nivo by SEC after combination of metal, peroxide and optical stress with thermal stress at 30 ℃ in study 1. RT/light is continuous light stress, other light stress conditions last for 3 days duration. Formulations 1 (air) and 6 (nitrogen) had: 50 μm DTPA, 5mM Met; formulations 2 (air) and 7 (nitrogen) have: 0 μM DTPA, 0mM Met. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0, and 2,000U/mL rHuPH20.

Fig. 11 is a graphical representation of the effect of various stress conditions on the% HMW species for Nivo by SEC after the combination of metal, peroxide and optical stress with thermal stress at 30 ℃ in study 1. RT/light is continuous light stress, other light stress conditions last for 3 days duration. Formulation 1:50 μm DTPA, 5mM Met; formulation 2:0 μM DTPA, 0mM Met; formulation 3:0 μM DTPA, 5mM Met; formulation 4: 50. Mu.M DTPA, 0mM Met; formulation 5: 100. Mu.M EDTA, 5mM Met. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0, and 2,000U/mL rHuPH20.

FIG. 12 is a graphical representation of HMW formation under metal (0.5 ppm each of iron, chromium and copper) +illumination (3 days at 1000 lux and room temperature +1mM peroxide +30℃ thermal stress) in study 1. Note that: formulations 1 (air) and 6 (nitrogen) were completely superimposed on top of each other and had minimal HMW formation for the same formulation composition. Formulations 1 (air) and 6 (nitrogen): 50 μm DTPA, 5mM Met; formulations 2 (air) and 7 (nitrogen): 0 μM DTPA, 0mM Met; formulation 3:0 μM DTPA, 5mM Met; formulation 4: 50. Mu.M DTPA, 0mM Met; formulation 5: 100. Mu.M EDTA, 5mM Met. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0, and 2,000U/mL rHuPH20.

FIG. 13 is a graphical representation of HMW% in study 1 after 3 months of various combinations of metals (0.5 ppm each of iron, chromium and copper), light (3 days at 1000 lux and room temperature) and peroxide (1 mM peroxide) with 30℃thermal stress, as formulated with/without 5mM Met and 50. Mu.M DTPA and 100. Mu.m EDTA. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0, and 2,000U/mL rHuPH20.

FIG. 14 is a graphical representation of the% HMW included in the primary statistical model of action after 3 months of thermal stress at 30℃in study 1, various combinations of metals (0.5 ppm each of iron, chromium and copper), light (3 days at 1000 lux and room temperature) and peroxide (1 mM peroxide). The pattern was divided according to the formulation composition with/without 5mM Met and 50. Mu.M DTPA. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0, and 2,000U/mL rHuPH20.

FIG. 15 is a graphical representation of rHuPH20 enzyme activity after 30℃in storage at 30℃in the dark after 3 days RT/darkness [ control-left panels ], 30℃in the dark after 3 days RT/RL and metal-and peroxide-labeled storage [ MPL-middle panels ] and storage at room temperature/room illumination [ RT/light-right panels ]. Formulations 1 (air) and 6 (nitrogen): 50 μm DTPA, 5mM Met; formulations 2 (air) and 7 (nitrogen): 0 μM DTPA, 0mM Met; formulation 3:0 μM DTPA, 5mM Met; formulation 4: 50. Mu.M DTPA and 0mM Met. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0, and 2,000U/mL rHuPH20.

Fig. 16A-16F are graphical representations of formulation profiles for study 2. The formulation ranges from max and min for the excipients, all other factors being at the following target composition: 120mg/mL Nivo (FIG. 16A), 20mM His (FIG. 6C), 250mM sucrose, 50 μM DTPA (FIG. 16D), 5mM Met (FIG. 16E), 2,000U/mL rHPH20 (FIG. 16F), 0.05% w/v PS80, pH 6.0 (FIG. 16B).

FIG. 17 is a graphical representation of high molecular weight species by SEC at various time points up to 6 months for study 2, for 0-200. Mu.M DTPA, for 25 ℃, 35 ℃ and MPL and RT/RL stress conditions. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 5mM Met, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

FIG. 18 is a graphical representation of the high molecular weight species by SEC at various time points up to 6 months for the formulations DTPA and Met concentration separations for study 2, for 25 ℃, 35 ℃ and MPL and RT/RL stress conditions. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

Figure 19 is a graphical representation of the high molecular weight species by SEC at various time points up to 6 months for study 2, separated by formulation DTPA and Met concentrations. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

FIG. 20 is a graphical representation of SEC HMW% versus methionine concentration at three last sampled (last-sampled) stress conditions and smooth curve trend estimates at the following compositions: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

Figures 21A-21C are graphical representations of linear regression models of total HMW% as a function of Met level after 6 months at 25 ℃ (figure 21A), 3 months at 35 ℃ (figure 21B) and 3 months under MPL stress (figure 21C), and fitted performance of formulations containing: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

Fig. 22A-22B are graphical representations of acidic species as a function of time under MPL conditions (fig. 22A) and stress at 35 ℃ (fig. 22B). Formulations with DTPA and Met, and multiple samples of DTPA alone. DTPA at 50. Mu.M and 5mM Met concentration. The formulation had 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2,000U/mL rHuPH20, pH 6.0.

FIGS. 23A-23B are graphical representations of enzyme activity as a function of time under MPL conditions (FIG. 23A) and stress at 35 ℃. Formulations with DTPA and Met, and multiple samples of DTPA alone. DTPA at 50. Mu.M and 5mM Met concentration. The formulation had 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2,000U/mL rHuPH20, pH 6.0.

Fig. 24A-24B are graphical representations of PS80 levels over time under MPL conditions (fig. 24A) and 35 ℃ stress (fig. 24B). Formulations with DTPA and Met, and multiple samples of DTPA alone. DTPA at 50. Mu.M and 5mM Met concentration. The formulation had 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2,000U/mL rHuPH20, pH 6.0.

FIG. 25A is a graphical representation illustrating a comparison between study 1, study 2 and study 3 for high molecular weight species by SEC at 3 month time points under MPL conditions with and without 2,000U/mL rHuPH20 enzyme separation at various Met levels. Fig. 25B is a regression plot of Met-dependent study 1, study 2 and study 3 for high molecular weight species by SEC at the 3 month time point of MPL conditions. The composition included 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0 (FIGS. 25A-25B).

FIG. 26 is a bar graph providing log for glycine, mannitol, sucrose, trehalose and succinate ₁₀ Comparison of (kd) as shown.

Figure 27 is a bar graph showing the average count of the number of excipient molecules that interacted with the nano Wu Shankang Fab group during the last 8ns of the MD simulation for glycine, sorbitol, trehalose, mannitol and sucrose, as shown.

Fig. 28A-28E are illustrations of the binding poses found on the nivolumab Fab for each of glycine (fig. 28A), sorbitol (fig. 28B), mannitol (fig. 28C), sucrose (fig. 28D), and trehalose (fig. 28E). Fab groups are shown as bands, light binding poses are shown in a club representation, and tight binding poses are shown in a space-filling representation.

Fig. 29A-29B are bar graphs illustrating the number of unique binding poses found in the MD simulation for each excipient (glycine, sorbitol, trehalose, mannitol, and sucrose) for medium strength interactions (fig. 29A) and strong binding interactions (fig. 29B).

Detailed Description

Current methods of delivering anti-PD-1 and/or anti-PD-L1 antibodies require periodic intravenous administration, typically by a clinician, typically at a clinic or hospital. This approach often causes significant inconvenience to the patient, and the nature of the treatment itself may negatively impact the patient's experience. Subcutaneous delivery (e.g., through the use of an auto-injector or wearable pump) can significantly improve patient compliance, potentially allowing the patient to comfortably receive such potentially life-saving therapy in his home. The present disclosure provides a method of treating a subject in need thereof, the method comprising subcutaneously administering to the subject a dose of a pharmaceutical composition comprising (i) an antibody that specifically binds PD-1 or PD-L1 and inhibits the interaction of PD-1 with PD-L1 (an "anti-PD-1 antibody" or an "anti-PD-L1 antibody", respectively). In some aspects, the pharmaceutical composition further comprises (ii) an endoglycosidase hydrolase. In some aspects, the dose comprises one or more subcutaneous unit doses.

In some aspects, the pharmaceutical composition does not comprise an endoglycosidase hydrolase. In some aspects, the total volume of at least one of the subcutaneous unit doses is less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 3mL, or less than about 2.5 mL). In certain aspects, the dose comprises at least about 250mg to at least about 2400mg of the antibody.

I. Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used herein, each of the following terms shall have the meanings set forth below, unless the context clearly provides otherwise. Additional definitions are set forth throughout this disclosure.

It should be understood that wherever aspects are described herein by the language "comprising," other similar aspects are also provided that are described in a manner that "consists of … …" and/or "consists essentially of … ….

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, concise Dictionary of Biomedicine and Molecular Biology, juo, pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3 rd edition, 1999, academic press; and Oxford Dictionary of Biochemistry And Molecular Biology, revisions, 2000, oxford university press, provide those skilled in the art with a general explanation of many of the terms used in this disclosure.

Units, prefixes, and symbols are all expressed in terms of their international units System (SI) acceptability. Numerical ranges include numbers defining the ranges. Unless otherwise indicated, nucleotide sequences are written in the 5 'to 3' direction from left to right. The amino acid sequence is written in the amino to carboxyl direction from left to right. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole. Accordingly, by referring to the specification in general, the terms defined immediately below are more fully defined.

"administering" refers to physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Administration may refer to any form of administration for immunotherapy (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies), including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion. The phrases "subcutaneously administering" and "subcutaneously administering" are used interchangeably and refer to a mode of administration in which a substance (e.g., a composition comprising an antibody that specifically binds PD-1 or PD-L1 and inhibits the interaction of PD-1 with PD-L1) is delivered to a subject under the skin, between the dermis and, for example, the muscle.

Subcutaneous administration may be accomplished using any method. In some aspects, subcutaneous administration is achieved using a short needle or a plurality of short needles. In some aspects, the needle or at least one of the plurality of needles is less than about 1 inch, less than about 7/8 inch, less than about 6/8 inch, less than about 5/8 inch, less than about 1/2 inch. In some aspects, the length of the needle or at least one of the plurality of needles is about 5/8 inch.

The administration may be performed, for example, one time, multiple times, and/or over one or more extended periods of time. Thus, as used herein, administration may refer to a single unit dose or more than one unit dose.

As used herein, the term "dose" or "dose" is defined as the amount of a therapeutic agent that can be administered at a given point. The dose or doses (dosage) may be an amount sufficient to achieve or at least partially achieve the desired effect, but such desired effect may not be visible or detectable. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any of the following amounts of the drug: when used alone or in combination with another therapeutic agent, promotes disease regression, manifested by a decrease in severity of disease symptoms, an increase in the frequency and duration of disease-free symptomatic periods, an increase in overall survival (the length of time that a patient diagnosed with a disease remains alive from the date of diagnosis of the disease or the date of starting treatment), or prevention of injury or disability due to disease affliction. The amount or dose of the drug includes a "prophylactically effective amount" or "prophylactically effective dose" which is any of the following amounts of the drug: when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or developing a recurrence of a disease, inhibits the progression or recurrence of the disease. The ability of a therapeutic agent to promote regression of a disease or inhibit progression or recurrence of a disease can be assessed using a variety of methods available to skilled practitioners, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in an in vitro assay. The "dose" may include a single unit dose or multiple unit doses. In some aspects, the dose comprises a single unit dose. In some aspects, the dose comprises a plurality of unit doses.

As used herein, a subcutaneous "unit dose" refers to a single amount of a substance delivered by subcutaneous injection (e.g., from a single vial, a single auto-injector, and/or a single syringe). In some aspects, multiple subcutaneous doses are administered to achieve a therapeutically effective dose. Where multiple unit doses are administered, the individual unit doses may be administered simultaneously or sequentially. In some aspects, each unit dose of the therapeutically effective dose is administered on the same day. Each unit dose may be administered at the same body location or at a different body location. In some aspects, the first unit dose is administered at a first body site and the second unit dose is administered at a second body site. Any body location known in the art to be suitable for subcutaneous delivery may be used in the methods disclosed herein. In some aspects, at least one subcutaneous unit dose of the doses is administered to a body site selected from the group consisting of: arms (e.g., side or rear of upper arm), abdomen, and thigh front.

As used herein, an "adverse event" (AE) is any adverse and often unintended or undesired sign (including abnormal laboratory findings), symptom, or disease associated with the use of medical treatment. For example, an adverse event may be associated with activation of the immune system or expansion of immune system cells (e.g., T cells) in response to treatment. Medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity. References to methods capable of "altering an adverse event" mean a treatment regimen that reduces the incidence and/or severity of one or more AEs associated with the use of different treatment regimens.

An "antibody" (Ab) shall include, but is not limited to, glycoprotein immunoglobulins which specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V _H ) And a heavy chain constant region. The heavy chain constant region comprises three constant domains, C _H1 、C _H2 And C _H3 . Each light chain comprises a light chain variable region (abbreviated herein as V _L ) And a light chain constant region. The light chain constant region comprises a constant domain, C _L 。V _H Region and V _L The regions may be further subdivided into regions of high denaturation called Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved, called Framework Regions (FR). Each V _H And V _L Comprising three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). Thus, the term "anti-PD-1 antibody" includes whole antibodies and antigen-binding portions of whole antibodies that specifically bind to PD-1, having two heavy chains and two light chains. Non-limiting examples of antigen binding moieties are shown elsewhere herein.

The immunoglobulin may be derived from any known isotype, including but not limited to IgA, secretory IgA, igG, and IgM. Subclasses of IgG are also well known to those of skill in the art and include, but are not limited to, human IgG1, igG2, igG3, and IgG4. "isotype" refers to the class or subclass of antibodies (e.g., igM or IgG 1) encoded by the heavy chain constant region gene. For example, the term "antibody" includes both naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric and humanized antibodies; a human antibody or a non-human antibody; fully synthesizing an antibody; a single chain antibody. The non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly indicated otherwise by context, the term "antibody" also includes antigen binding fragments or antigen binding portions of any of the above immunoglobulins, and includes monovalent and bivalent fragments or portions as well as single chain antibodies.

In some aspects, an "antibody" of the disclosure is capable of binding to more than one antigen, e.g., a "multispecific" antibody. As used herein, a "bispecific" antibody is an antibody capable of specifically binding two antigens, wherein the first antigen and the second antigen are the same or different. As used herein, a "multispecific" antibody is capable of specifically binding more than one antigen, e.g., at least two (i.e., a "bispecific" antibody), at least three (i.e., a "trispecific" antibody), at least four, at least five, or at least six antigens. A variety of multispecific antibodies are known and may be used in the compositions and/or methods disclosed herein, including, but not limited to, bispecific antibodies that bind PD-1 and a second target and bispecific antibodies that bind PD-L1 and a second target. In some aspects, the multispecific antibody is a T cell-dependent bispecific antibody. In some aspects, the multispecific antibody is an anti-FcRH 5/CD3 bispecific antibody targeting the B cell lineage markers FcRH5 and CD3, e.g., for use in the treatment of multiple myeloma.

In some aspects, an "antibody" of the disclosure is engineered to activate at a target site, e.g., a "pre-antibody (probody)". In some aspects, the antibody (e.g., a pre-antibody) is proteolytically cleaved at the target tissue (e.g., tumor).

An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, isolated antibodies that specifically bind to PD-1 may have cross-reactivity with other antigens (e.g., PD-1 molecules from different species). Furthermore, the isolated antibodies may be substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of antibody molecules having a single molecular composition, i.e., antibody molecules whose primary sequences are substantially identical and exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are examples of isolated antibodies. Monoclonal antibodies may be produced by hybridomas, recombination, transgenes, or other techniques known to those skilled in the art.

"human antibody" (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains constant regions, the constant regions are also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

"humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDRs of a non-human antibody have been replaced by corresponding amino acids derived from a human immunoglobulin. In one aspect of the humanized form of the antibody, some, most or all of the amino acids outside of the CDRs have been replaced by amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDRs have not been altered. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible provided they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized antibodies" retain antigen specificity similar to the original antibody.

"chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

An "anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-PD-L1 antibody specifically binds to PD-L1.

An "antigen-binding portion" of an antibody (also referred to as an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen to which the intact antibody binds. It has been shown that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody as described herein) include (i) Fab fragments (fragments from papain cleavage) or V _L 、V _H Similar monovalent fragments consisting of LC and CH1 domains; (ii) F (ab') 2 fragments (fragments from pepsin cleavage) or similar bivalent fragments comprising two Fab fragments linked by a disulfide bridge of a hinge region; (iii) From V _H And a CH1 domain; (iv) V by antibody single arm _L And V _H Fv fragments consisting of domains; (v) dAb fragment (Ward et al, (1989) Nature 341:544-546) whichFrom V _H Domain composition; (vi) an isolated Complementarity Determining Region (CDR); and (vii) a combination of two or more isolated CDRs which may optionally be linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment V _L And V _H Encoded by separate genes, but can be joined by synthetic linkers using recombinant methods, enabling them to be made into a single protein chain in which the V _L Region and the V _H The pairing of regions forms monovalent molecules, known as single chain Fv (scFv); see, e.g., bird et al (1988) Science 242:423-426; huston et al (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for utility in the same manner as whole antibodies. The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

"cancer" refers to a broad group of different diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distal parts of the body through the lymphatic system or blood flow.

The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of infection or recurrence of a disease by a method comprising inducing, enhancing, suppressing or otherwise altering an immune response. "treatment" or "therapy" of a subject refers to any type of intervention or treatment performed on the subject, or administration of an active agent to the subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing or preventing the onset, progression, development, severity or recurrence of symptoms, complications or disorders, or biochemical indicators associated with the disease.

"programmed death protein-1" (PD-1) refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed primarily on previously activated T cells in vivo and binds to two ligands, namely PD-L1 and PD-L2. As used herein, the term "PD-1" includes variants, isoforms and species homologs of human PD-1 (hPD-1), hPD-1, and analogs having at least one common epitope with hPD-1. Complete hPD-1 sequences can be found under GenBank accession number U64863.

"programmed death protein ligand-1" (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which down-regulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. Complete hPD-L1 sequences can be found under GenBank accession number Q9 NZQ. The human PD-L1 protein is encoded by the human CD274 gene (NCBI gene ID: 29126).

As used herein, "hyaluronidase" refers to an enzyme capable of catalyzing cleavage of hyaluronic acid. Hyaluronic acid is a repeating polymer of N-acetyl-glucosamine and glucuronic acid, which is present in the subcutaneous space and contributes to the soluble gel-like components of the extracellular matrix of the skin and is recovered by rapid turnover (resynthesis). In some aspects, the hyaluronidase comprises rHuPH20, which is a glycosylated 447 amino acid single-chain polypeptide that locally depolymerizes hyaluronic acid in the subcutaneous space at the injection site of the skin. Depolymerization of hyaluronic acid by hyaluronidase is accomplished by hydrolysis of the polysaccharide polymer. The depolymerization of hyaluronic acid results in a short decrease in viscosity of the gel-like phase of the extracellular matrix and increases moisture conductivity, thereby promoting dispersion and absorption of co-administered therapeutic agents. Thus, hyaluronidase (e.g., rHuPH 20) can improve the speed and ease of subcutaneous delivery of injectable biologics and drugs by acting as a permeation enhancer. In certain aspects, the hyaluronidase comprises ENHANZE ^TM 。

"subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents (e.g., mice, rats, and guinea pigs). In a preferred aspect, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

The use of the term "flat dose" in relation to the methods and doses of the present disclosure means a dose administered to a patient irrespective of the patient's body weight or Body Surface Area (BSA). Thus, the flat dose is not provided in mg/kg doses, but in absolute amounts of the agent (e.g., anti-PD-1 antibody). For example, 60kg of a human and 100kg of a human will receive the same dose of antibody (e.g., 240mg of anti-PD-1 antibody).

The term "weight-based dose" as referred to herein means the dose administered to a patient calculated based on the weight of the patient. For example, when a patient weighing 60kg requires 3mg/kg of anti-PD-1 antibody, one can calculate and administer an appropriate amount of anti-PD-1 antibody (i.e., 180 mg).

For example, an "anticancer agent" promotes cancer regression in a subject. In some aspects, a therapeutically effective amount of the drug promotes regression of the cancer to the point of eliminating the cancer. By "promoting cancer regression" is meant that administration of a therapeutically effective amount of the drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of disease-free symptomatic periods, or prevention of injury or disability due to disease affliction. In addition, the terms "effective" and "effectiveness" with respect to treatment include pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote regression of a patient's cancer. Physiological safety refers to the level of toxicity caused by administration of a drug or other adverse physiological effects (adverse effects) at the cellular, organ and/or organism level.

For example, for the treatment of a tumor, a therapeutically effective amount of the anti-cancer agent preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to an untreated subject. In other preferred aspects of the present disclosure, tumor regression may be observed and continued for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days. Despite the final measurement of the effectiveness of these treatments, the evaluation of immunotherapeutic drugs must also take into account immune-related response patterns.

An "immune response" is understood in the art and generally refers to a biological response within a vertebrate against foreign factors (agents) or abnormalities such as cancer cells that protect the organism from these factors and diseases caused thereby. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in selective targeting, binding, damage, destruction, and/or elimination of an invading pathogen, pathogen-infected cell or tissue, cancerous or other abnormal cell in the vertebrate body, or in the case of autoimmune or pathological inflammation, selective targeting, binding, damage, destruction, and/or elimination of normal human cells or tissue. The immune response includes, for example, T cells (e.g., effector T cells, th cells, CD4 ⁺ Cell, CD8 ⁺ T cells or Treg cells), or activation or inhibition of any other cell in the immune system (e.g., NK cells).

By "immune-related response pattern" is meant the clinical response pattern typically observed in cancer patients treated with immunotherapeutic agents that produce an anti-tumor effect by inducing a cancer-specific immune response or by modifying the innate immune process. This response pattern is characterized by a beneficial therapeutic effect after an initial increase in tumor burden or the appearance of new lesions, which will be classified as disease progression in the evaluation of traditional chemotherapeutic agents and will be synonymous with drug failure. Thus, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effect of these agents on the disease of interest.

As used herein, the term "stable" with respect to a formulation or pharmaceutical product is a term in which one or more antibodies or molecules substantially retain their physical and chemical stability and integrity after storage. Stability of the formulations herein can be measured at a selected temperature after a selected period of time. For example, aggregate formation or an increase in low molecular weight species is an indicator of instability. The maintenance of initial clarity and/or color over the shelf life is also an indicator for monitoring stability. In some aspects, a "stable" drug product is one in which the increase in aggregation, as measured by the percentage increase in high molecular weight species (HMW%) is less than about 5%, preferably less than about 3%, upon storage of the formulation at 2-8 ℃ for at least about one year.

As used herein, the terms "treatment", "treatment" and "treatment" refer to any type of intervention or procedure performed on a subject with the purpose of reversing, alleviating, inhibiting or slowing or preventing the progression, development, severity or recurrence of symptoms, complications, disorders or biochemical indicators associated with a disease, or increasing overall survival. The treatment may be treatment (e.g., for prophylaxis) of a subject with a disease or a subject without a disease.

The terms "about weekly", "about biweekly", or any other similar dosing interval terms as used herein mean approximations. "about weekly" may include every seven days + -one day, i.e., every six days to every eight days. "about every two weeks" may include every fourteen days ± three days, i.e., every ten days to every seventy days. Similar approximations apply, for example, about every three weeks, about every four weeks, about every five weeks, about every six weeks, and about every twelve weeks. In some aspects, dosing intervals of about every six weeks or about every twelve weeks means that a first dose may be administered on any of the first week, and then a next dose may be administered on any of the sixth or twelfth weeks, respectively. In other aspects, a dosing interval of about every six weeks or about every twelve weeks means that a first dose is administered on a particular day of the first week (e.g., monday) and then the next dose is administered on the same day of the sixth or twelfth week (i.e., monday), respectively. When multiple subcutaneous unit doses are administered to achieve a dose, the dosing interval refers to the period of time between the administration of a first subcutaneous unit dose of a first effective dose and the administration of a first subcutaneous unit dose of a second effective dose. For example, where the method comprises administering a dose of about 600mg administered about every two weeks, wherein the dose of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 300mg of the antibody, the first subcutaneous unit dose of about 300mg of the first effective dose of the antibody is administered on day 1, and the first subcutaneous unit dose of about 300mg of the second effective dose of the antibody is administered on day 14. In this example, a second unit dose of about 300mg of the first effective dose of the antibody may be administered on day 1 or any other time prior to administration of the first subcutaneous unit dose of about 300mg of the second effective dose of the antibody.

The use of alternatives (e.g., "or") should be understood to mean either, both, or any combination thereof. As used herein, the indefinite article "a" or "an" is to be understood to mean "one or more" of any recited or enumerated component.

The term "about" or "consisting essentially of … …" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" or "consisting essentially of … …" may mean within 1 or more than 1 standard deviation. Alternatively, "about" or "consisting essentially of … …" may mean a range of up to 10%. Furthermore, in particular with respect to biological systems or processes, the term may mean at most one order of magnitude or at most 5 times the value. When a particular value or composition is provided in the application and claims, unless otherwise indicated, it should be assumed that the meaning of "about" or "consisting essentially of … …" is within an acceptable error of that particular value or composition.

As described herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer and (where appropriate) fractional (e.g., one tenth and one hundredth) values of any integer within the recited range.

Various aspects of the disclosure are described in more detail in the following subsections.

Methods of the present disclosure

Some aspects of the disclosure relate to methods of treating a subject in need thereof, the methods comprising subcutaneously administering to the subject a dose of a pharmaceutical composition comprising (i) an antibody that specifically binds PD-1 or PD-L1 and inhibits the interaction of PD-1 with PD-L1 (an "anti-PD-1 antibody" or an "anti-PD-L1 antibody", respectively). In some aspects, the pharmaceutical composition comprises (ii) an endoglycosidase hydrolase. In some aspects, the dose may be a therapeutically effective dose. In some aspects, the dose comprises one or more subcutaneous unit doses. In some aspects, the dose comprises at least about 300mg to at least about 2400mg of the anti-PD-1 antibody or the anti-PD-L1 antibody.

In some aspects, the pharmaceutical composition does not comprise an endoglycosidase hydrolase. In some aspects, the total volume of at least one of the subcutaneous unit doses is less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 3mL, or less than about 2.5 mL). In certain aspects, the therapeutically effective dose comprises at least about 250mg to at least about 2400mg of the antibody. In some aspects, the pharmaceutical composition does not comprise an enzyme that facilitates subcutaneous delivery. In certain aspects, the pharmaceutical composition does not comprise hyaluronidase.

II.A. administration

In some aspects, the therapeutically effective dose of the antibody comprises a single subcutaneous unit dose, e.g., the entire dose is administered as a single unit dose. In some aspects, the therapeutically effective dose of the antibody comprises two or more subcutaneous unit doses, e.g., the effective unit dose is divided into two or more subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least two subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least three subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least four subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least five subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least six subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least seven subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least eight subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises at least nine subcutaneous unit doses. In some aspects, the therapeutically effective dose of the antibody comprises ten or more subcutaneous unit doses.

In some aspects, each subcutaneous unit dose is administered on the same day. In some aspects, one or more subcutaneous unit doses are administered on a first day and one or more subcutaneous unit doses of the same therapeutically effective dose are administered on a second day. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered sequentially. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose of the same effective dose are administered sequentially, wherein the second subcutaneous unit dose is administered less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes, less than about 45 minutes, less than about 60 minutes, less than about 75 minutes, less than about 90 minutes, less than about 2 hours, less than about 2.5 hours, less than about 3 hours, less than about 3.5 hours, less than about 4 hours, less than about 4.5 hours, less than about 5 hours, less than about 5.5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 12 hours, less than about 18 hours, or less than about 24 hours after the first subcutaneous unit dose. In some aspects, the two or more subcutaneous unit doses are administered sequentially, wherein each of the two or more subcutaneous unit doses is administered within an interval of less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes, less than about 45 minutes, less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 15 hours, less than about 18 hours, less than about 21 hours, or less than about 24 hours between the subcutaneous unit doses.

In some aspects, the one or more subcutaneous unit doses are administered at one or more body locations. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at the same body location. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site and a second body site, wherein the first body site is different from the second body site. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body location and the third subcutaneous unit dose is administered at a second body location, wherein the first body location is different from the second body location. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body location and the third subcutaneous unit dose and the fourth subcutaneous dose are administered at a second body location, wherein the first body location is different from the second body location. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, and the third subcutaneous unit dose is administered at a third body site, wherein the first body site, the second body site, and the third body site are different. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, and the fourth subcutaneous unit dose is administered at a fourth body site, wherein the first body site, the second body site, the third body site, and the fourth body site are different.

As used herein, where at least two subcutaneous unit doses are administered at the same body location, the two subcutaneous doses may be administered at the exact same injection site or at nearby injection sites within the same body location. For example, two subcutaneous doses administered to a single body site may both be administered to the right arm of the subject. In this example, as long as two subcutaneous doses are administered to, for example, the right arm, then two subcutaneous unit doses are administered to the same "body site," as used herein.

The therapeutically effective dose and/or subcutaneous dose may be administered subcutaneously as disclosed herein using any method or device. In some aspects, the therapeutically effective dose and/or subcutaneous dose is administered using a syringe. In some aspects, the therapeutically effective dose and/or subcutaneous dose is administered using an automatic injector. In some aspects, the therapeutically effective dose and/or subcutaneous dose is administered using an injection pen. In some aspects, the therapeutically effective dose and/or the subcutaneous dose is administered using a wearable pump.

In some aspects, the therapeutically effective dose and/or subcutaneous unit dose is administered by subcutaneous infusion for less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, less than about 15 minutes, less than about 14 minutes, less than about 13 minutes, less than about 12 minutes, less than about 11 minutes, less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, or less than about 2 minutes. In some aspects, the therapeutically effective dose and/or subcutaneous unit dose is administered by subcutaneous infusion for less than about 90 seconds, less than about 75 seconds, less than about 60 seconds, less than about 45 seconds, less than about 30 seconds, less than about 15 seconds, or less than about 10 seconds. In some aspects, the therapeutically effective dose and/or subcutaneous unit dose is administered by subcutaneous infusion for less than about 15 minutes. In some aspects, the therapeutically effective dose and/or subcutaneous unit dose is administered by subcutaneous infusion for less than about 10 minutes. In some aspects, the therapeutically effective dose and/or subcutaneous unit dose is administered by subcutaneous infusion for less than about 5 minutes. In some aspects, the therapeutic dose and/or subcutaneous dose is administered by subcutaneous infusion for less than about 4 minutes. In some aspects, the therapeutically effective dose and/or the subcutaneous dose is administered by subcutaneous infusion for less than about 3 minutes. In some aspects, the therapeutically effective dose and/or subcutaneous unit dose is administered by subcutaneous infusion for less than about 2 minutes.

II.A.1. administration of anti-PD-1 antibodies

In certain aspects of the disclosure, the antibodies comprise anti-PD-1 antibodies. Any anti-PD-1 antibody can be used in the methods disclosed herein. In some aspects, the anti-PD-1 antibody comprises nivolumab. In some aspects, the anti-PD-1 antibody comprises pembrolizumab.

In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 250mg to about 600mg of the antibody per week. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 250mg to about 550mg, about 250mg to about 500mg, about 250mg to about 450mg, about 250mg to about 400mg, about 250mg to about 350mg, about 250mg to about 300mg, about 275mg to about 400mg, about 275mg to about 375mg, about 275mg to about 350mg, about 275mg to about 325mg, about 275mg to about 300mg, about 300mg to about 600mg, about 300mg to about 550mg, about 300mg to about 400mg, about 300mg to about 450mg, about 300mg to about 400mg, about 300mg to about 350mg, or about 300mg to about 325mg of the antibody per week. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 250mg to about 400mg of the antibody per week. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 250mg to about 350mg of the antibody per week. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 275mg to about 325mg of the antibody per week.

In some aspects, the antibody is administered at a dose of about 250mg, about 260mg, about 270mg, about 275mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 325mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 375mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 425mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 475mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 525mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 575mg, about 580mg, about 590mg, or about 600mg per week. In certain aspects, the antibody is administered at a dose of about 250mg per week. In certain aspects, the antibody is administered at a dose of about 275mg per week. In certain aspects, the antibody is administered at a dose of about 300mg per week. In certain aspects, the antibody is administered at a dose of about 325mg per week. In certain aspects, the antibody is administered at a dose of about 350mg per week.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 300mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 300mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 300mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 150mg of the antibody. In some aspects, at least one of the two subcutaneous unit doses comprises less than about 150mg of the antibody in a total volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, the two subcutaneous unit doses are administered to two different body locations of the subject.

In some aspects, the dose comprises three subcutaneous unit doses, wherein each of the three subcutaneous unit doses comprises about 100mg of the antibody. In some aspects, at least one of the three subcutaneous unit doses comprises about 100mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the three subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site and the third subcutaneous unit dose is administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, and the third subcutaneous unit dose is administered at a third body site.

In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 300mg to about 900mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 300mg to about 850mg, about 300mg to about 800mg, about 300mg to about 750mg, about 300mg to about 700mg, about 300mg to about 650mg, about 300mg to about 600mg, about 350mg to about 900mg, about 350mg to about 850mg, about 350mg to about 800mg, about 350mg to about 750mg, about 350mg to about 700mg, about 350mg to about 650mg, about 350mg to about 600mg, about 400mg to about 900mg, about 400mg to about 850mg, about 400mg to about 800mg, about 400mg to about 750mg, about 400mg to about 700mg, about 400mg to about 650mg, about 400mg to about 600mg, about 450 to about 900mg, about 450 to about 850mg, about 450 to about 800mg, about 450mg to about 750mg about 450mg to about 700mg, about 450mg to about 650mg, about 450mg to about 600mg, about 500mg to about 900mg, about 500mg to about 850mg, about 500mg to about 800mg, about 500mg to about 700mg, about 500mg to about 650mg, about 500mg to about 600mg, about 550mg to about 900mg, about 550mg to about 850mg, about 550mg to about 800mg, about 550mg to about 750mg, about 550mg to about 700mg, about 550mg to about 650mg, about 550mg to about 600mg, about 600mg to about 900mg, about 600mg to about 850mg, about 600mg to about 800mg, about 600mg to about 750mg, about 600mg to about 700mg, about 600mg to about 650mg, about 575mg to about 625mg, about 575mg to about 600mg, or about 600mg to about 625 mg. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 400mg to about 800mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 500mg to about 700mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 550mg to about 650mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 575mg to about 625mg of the antibody every two weeks.

In some aspects, the antibody is administered at a dose of about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 730mg, about 740mg, about 750mg, about 760mg, about 770mg, about 780mg, about 790mg, about 800mg, about 810mg, about 820mg, about 830mg, about 840mg, about 850mg, about 860mg, about 870mg, about 880mg, about 890mg, or about 900mg every two weeks. In some aspects, the antibody is administered at a dose of about 500mg every two weeks. In some aspects, the antibody is administered at a dose of about 550mg every two weeks. In some aspects, the antibody is administered at a dose of about 575mg every two weeks. In some aspects, the antibody is administered at a dose of about 600mg every two weeks. In some aspects, the antibody is administered at a dose of about 625mg every two weeks. In some aspects, the antibody is administered at a dose of about 650mg every two weeks. In some aspects, the antibody is administered at a dose of about 700mg every two weeks.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600 mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 600mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 600mg in a total administration volume of about 5 mL.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600mg in a total administered volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL).

In some aspects, the dosage of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 300mg of the antibody. In some aspects, at least one of the two subcutaneous unit doses comprises about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the two subcutaneous unit doses comprises less than about 300mg of the antibody in a total volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In certain aspects, at least one of the two subcutaneous unit doses comprises less than about 300mg of the antibody in a total volume of about 2 mL. In some aspects, the two subcutaneous unit doses are administered to a single body location of the subject. In some aspects, the two subcutaneous unit doses are administered to two different body locations of the subject.

In some aspects, a dose of about 600mg of the antibody comprises three subcutaneous unit doses. In some aspects, at least one of the three subcutaneous unit doses comprises about 200mg of the antibody. In some aspects, each of the three subcutaneous unit doses comprises about 200mg of the antibody. In some aspects, at least one of the three subcutaneous unit doses comprises about 200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 200mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the three subcutaneous unit doses comprises about 200mg of the antibody in a total volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the three subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site and the third subcutaneous unit dose is administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, and the third subcutaneous unit dose is administered at a third body site.

In some aspects, a dose of about 600mg of the antibody comprises at least four subcutaneous unit doses. In some aspects, at least one of the four subcutaneous unit doses comprises about 150mg of the antibody. In some aspects, at least one of the four subcutaneous unit doses comprises about 150mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 150mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 150mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the four subcutaneous unit doses comprises less than about 150mg of the antibody in a total volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the four subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, and the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, and the fourth subcutaneous unit dose is administered at a fourth body site.

In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 900mg to about 1500mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 900mg to about 1450mg, about 900mg to about 1400mg, about 900mg to about 1350mg, about 900mg to about 1300mg, about 900mg to about 1250mg, about 900mg to about 1200mg, about 950mg to about 1500mg, about 950mg to about 1450mg, about 950mg to about 1400mg, about 950mg to about 1350mg, about 950mg to about 1300mg, about 950mg to about 1250mg, about 950mg to about 1200mg, about 1000mg to about 1500mg, about 1000mg to about 1450mg, about 1000mg to about 1400mg, about 1000mg to about 1350mg, about 1000mg to about 1300mg, about 1000mg to about 1250mg, about 1000mg to about 1200mg, about 1050mg to about 1500mg, about 1050 to about 1450mg, about 1050mg to about 1400mg, about 1050mg to about 1350mg, about 1050mg, about 1300mg about 1050mg to about 1250mg, about 1050mg to about 1200mg, about 1100mg to about 1500mg, about 1100mg to about 1450mg, about 1100mg to about 1400mg, about 1100mg to about 1350mg, about 1100mg to about 1300mg, about 1100mg to about 1250mg, about 1100mg to about 1200mg, about 1150mg to about 1500mg, about 1150mg to about 1450mg, about 1150mg to about 1400mg, about 1150mg to about 1350mg, about 1150mg to about 1300mg, about 1150mg to about 1250mg, about 1150mg to about 1200mg, about 1200mg to about 1500mg, about 1200mg to about 1450mg, about 1200mg to about 1400mg, about 1200mg to about 1350mg, about 1200mg to about 1300mg, about 1200mg to about 1250mg, about 1175mg to about 1225mg, about 1175mg to about 1200mg, or about 1200mg to about 1225mg of the antibody. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 1000mg to about 1400mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 1100mg to about 1300mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody) is administered at a dose of about 1150mg to about 1250mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 1175mg to about 1225mg of the antibody every four weeks.

In some aspects, the antibody is administered at a dose of about 900mg, about 950mg, about 1000mg, about 1010mg, about 1020mg, about 1030mg, about 1040mg, about 1050mg, about 1060mg, about 1070mg, about 1080mg, about 1090mg, about 1100mg, about 1110mg, about 1120mg, about 1130mg, about 1140mg, about 1150mg, about 1160mg, about 1170mg, about 1180mg, about 1190mg, about 1200mg, about 1210mg, about 1220mg, about 1230mg, about 1240mg, about 1250mg, about 1260mg, about 1270mg, about 1280mg, about 1300mg, about 1310mg, about 1320mg, about 1330mg, about 1340mg, about 1350mg, about 1360mg, about 1370mg, about 1470mg, about 1390mg, about 1400mg, about 1410mg, about 1420mg, about 1430mg, about 1440mg, about 1450mg, about 1460mg, about 1490mg, or about 1500mg. In some aspects, the antibody is administered at a dose of about 1100mg every four weeks. In some aspects, the antibody is administered at a dose of about 1150mg every four weeks. In some aspects, the antibody is administered at a dose of about 1175mg every four weeks. In some aspects, the antibody is administered at a dose of about 1200mg every four weeks. In some aspects, the antibody is administered at a dose of about 1225mg about every four weeks. In some aspects, the antibody is administered at a dose of about 1250mg every four weeks. In some aspects, the antibody is administered at a dose of about 1300mg every four weeks.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 1200 mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 1200mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 1200mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 1200mg in a total administration volume of about 5 mL.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of less than about 1200mg in a total administered volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL).

In some aspects, the dosage of the antibody comprises two, three, four, six, or at least eight subcutaneous unit doses. In some aspects, the dosage of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 600mg of the antibody. In some aspects, at least one of the two subcutaneous unit doses comprises about 600mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 600mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 600mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the two subcutaneous unit doses comprises about 600mg of the antibody in a total volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In certain aspects, at least one of the two subcutaneous unit doses comprises less than about 600mg of the antibody in a total volume of about 2 mL. In some aspects, the two subcutaneous unit doses are administered to a single body location of the subject. In some aspects, the two subcutaneous unit doses are administered to two different body locations of the subject.

In some aspects, a dose of about 1200mg of the antibody comprises three subcutaneous unit doses. In some aspects, at least one of the three subcutaneous unit doses comprises about 400mg of the antibody. In some aspects, each of the three subcutaneous unit doses comprises about 400mg of the antibody. In some aspects, at least one of the three subcutaneous unit doses comprises about 400mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 400mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 400mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the three subcutaneous unit doses comprises about 400mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the three subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site and the third subcutaneous unit dose is administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, and the third subcutaneous unit dose is administered at a third body site.

In some aspects, a dose of about 1200mg of the antibody comprises four subcutaneous unit doses. In some aspects, at least one of the four subcutaneous unit doses comprises about 300mg of the antibody. In some aspects, at least one of the four subcutaneous unit doses comprises about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the four subcutaneous unit doses comprises about 300mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the four subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, and the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, and the fourth subcutaneous unit dose is administered at a fourth body site.

In some aspects, a dose of about 1200mg of the antibody comprises six subcutaneous unit doses. In some aspects, at least one of the six subcutaneous unit doses comprises about 200mg of the antibody. In some aspects, at least one of the six subcutaneous unit doses comprises about 200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the six subcutaneous unit doses comprises greater than about 200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the six subcutaneous unit doses comprises greater than about 200mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the six subcutaneous unit doses comprises about 200mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the six subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site, and the fifth subcutaneous unit dose and the sixth subcutaneous unit dose are administered at a third body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, the fourth subcutaneous unit dose is administered at a fourth body site, the fifth subcutaneous unit dose is administered at a fifth body site, and the sixth subcutaneous unit dose is administered at a sixth body site.

In some aspects, a dose of about 1200mg of the antibody comprises at least eight subcutaneous unit doses. In some aspects, at least one of the eight subcutaneous unit doses comprises about 150mg of the antibody. In some aspects, at least one of the eight subcutaneous unit doses comprises about 150mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the eight subcutaneous unit doses comprises greater than about 150mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the eight subcutaneous unit doses comprises greater than about 150mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the eight subcutaneous unit doses comprises about 150mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the eight subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site, the fifth subcutaneous unit dose and the sixth subcutaneous unit dose are administered at a third body site, and the seventh subcutaneous unit dose and the eighth subcutaneous unit dose are administered at a fourth body site.

In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 1800mg to about 3000mg of the antibody every eight weeks. In some aspects, the antibody (e.g., the anti-PD-1 antibody (e.g., nivolumab)) is administered at a dose of about 1800mg to about 3000mg of the antibody every eight weeks. In some aspects of the present invention, the antibody is administered at a dose of about 1900mg, about 1950mg, about 2000mg, about 2010mg, about 2020mg, about 2030mg, about 2040mg, about 2050mg, about 2060mg, about 2070mg, about 2080mg, about 2090mg, about 2100mg, about 2110mg, about 2120mg, about 2130mg, about 2140mg, about 2150mg, about 2160mg, about 2170mg, about 2180mg, about 2190mg, about 2200mg, about 2210mg, about 2220mg, about 2230mg, about 2240mg, about 2250mg, about 2260mg, about 2270mg, about about 2280mg, about 2290mg, about 2300mg, about 2310mg, about 2320mg, about 2330mg, about 2340mg, about 2350mg, about 2360mg, about 2370mg, about 2380mg, about 2390mg, about 2400mg, about 2410mg, about 2420mg, about 2430mg, about 2440mg, about 2450mg, about 2460mg, about 2470mg, about 2480mg, about 2490mg, about 2500mg, about 2510mg, about 2520mg, about 2530mg, about 2540mg, about 2550mg, about 2560mg, about 2570mg, about 2580mg, about 2590mg or about 2600mg. In some aspects, the antibody is administered at a dose of about 2300mg every four weeks. In some aspects, the antibody is administered at a dose of about 2350mg every four weeks. In some aspects, the antibody is administered at a dose of about 2375mg every four weeks. In some aspects, the antibody is administered at a dose of about 2400mg every four weeks. In some aspects, the antibody is administered at a dose of about 2425mg every four weeks. In some aspects, the antibody is administered at a dose of about 2450mg about every four weeks. In some aspects, the antibody is administered at a dose of about 2475mg every four weeks. In some aspects, the antibody is administered at a dose of about 2500mg every four weeks.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 2400 mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 2400mg in a total administration volume of about 5 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 2400mg in a total administration volume of about 5 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 2400mg in a total administered volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL).

In some aspects, a dose of about 2400mg of the antibody comprises four subcutaneous unit doses. In some aspects, at least one of the four subcutaneous unit doses comprises about 600mg of the antibody. In some aspects, at least one of the four subcutaneous unit doses comprises about 600mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 600mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 600mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the four subcutaneous unit doses comprises about 600mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the four subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, and the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, and the fourth subcutaneous unit dose is administered at a fourth body site.

In some aspects, a dose of about 2400mg of the antibody comprises six subcutaneous unit doses. In some aspects, at least one of the six subcutaneous unit doses comprises about 400mg of the antibody. In some aspects, at least one of the six subcutaneous unit doses comprises about 400mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the six subcutaneous unit doses comprises greater than about 400mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the six subcutaneous unit doses comprises greater than about 400mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the six subcutaneous unit doses comprises about 400mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the six subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site, and the fifth subcutaneous unit dose and the sixth subcutaneous unit dose are administered at a third body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, the fourth subcutaneous unit dose is administered at a fourth body site, the fifth subcutaneous unit dose is administered at a fifth body site, and the sixth subcutaneous unit dose is administered at a sixth body site.

In some aspects, a dose of about 2400mg of the antibody comprises at least eight subcutaneous unit doses. In some aspects, at least one of the eight subcutaneous unit doses comprises about 300mg of the antibody. In some aspects, at least one of the eight subcutaneous unit doses comprises about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the eight subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the eight subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the eight subcutaneous unit doses comprises about 300mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the eight subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site, the fifth subcutaneous unit dose and the sixth subcutaneous unit dose are administered at a third body site, and the seventh subcutaneous unit dose and the eighth subcutaneous unit dose are administered at a fourth body site.

In some aspects, the anti-PD-1 antibody comprises pembrolizumab, which is administered subcutaneously, about once per week, about once every two weeks, about once every three weeks, or about once every four weeks. In some aspects, from about 100mg to about 300mg of pembrolizumab is administered subcutaneously, about once every two weeks. In some aspects, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, or about 300mg of pembrolizumab is administered subcutaneously, about once every two weeks. In some aspects, at least about 150mg of pembrolizumab is administered subcutaneously, about once every two weeks. In some aspects, at least about 200mg of pembrolizumab is administered subcutaneously, about once every two weeks. In some aspects, at least about 300mg of pembrolizumab is administered subcutaneously, about once every four weeks. In some aspects, at least about 400mg of pembrolizumab is administered subcutaneously, about once every four weeks. In some aspects, the dose of pembrolizumab is administered in a volume of at least about 2mL to at least about 4 mL.

In some aspects, the anti-PD-1 antibody comprises a sartoriab that is administered subcutaneously, about once a week, about once every two weeks, about once every three weeks, or about once every four weeks. In some aspects, about 200mg to about 400mg of sartoriab is administered subcutaneously, about once every four weeks. In some aspects, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, or about 400mg of sartoriab is administered subcutaneously, about once every four weeks. In some aspects, at least about 250mg of the sartoriab is administered subcutaneously, about once every four weeks. In some aspects, at least about 200mg of the sartoriab is administered subcutaneously, about once every four weeks. In some aspects, at least about 250mg of the sartoriab is administered subcutaneously, about once every four weeks. In some aspects, at least about 300mg of the sartoriab is administered subcutaneously, about once every four weeks. In some aspects, the dose of sartoriab is administered in a single injection in a volume of at least about 2 mL. In some aspects, the dose of sartoriab is administered in a volume of at least about 6mL in at least three injections.

In some aspects, the anti-PD-1 antibody comprises KN035, which is administered subcutaneously, about once per week, about once every two weeks, about once every three weeks, or about once every four weeks. In some aspects, about 100mg to about 200mg KN035 is administered subcutaneously, about once a week. In some aspects, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, or about 200mg KN035 is administered subcutaneously, about once a week. In some aspects, at least about 150mg KN035 is administered subcutaneously, about once a week. In some aspects, about 2.5mg/kg KN035 is administered subcutaneously, about once a week. In some aspects, about 200mg to about 400mg KN035 is administered subcutaneously, about once every three weeks. In some aspects, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, or about 400mg KN035 is administered subcutaneously about once every three weeks. In some aspects, at least about 300mg KN035 is administered subcutaneously, about once every three weeks. In some aspects, at least about 300mg KN035 is administered subcutaneously, about once every four weeks. In some aspects, at least about 400mg KN035 is administered subcutaneously, about once every four weeks. In some aspects, the dose of KN035 is administered in a volume of less than about 1 mL.

II.A.2. administration of anti-PD-L1 antibodies

In some aspects of the disclosure, the antibody comprises an anti-PD-L1 antibody. Any anti-PD-L1 antibody known in the art and/or disclosed herein may be used in the methods disclosed herein. In certain aspects, the anti-PD-L1 antibody comprises alemtuzumab. In certain aspects, the anti-PD-L1 antibody comprises cerulomumab. In certain aspects, the anti-PD-L1 antibody comprises avermectin.

In some aspects, the dosage of the antibody (e.g., the anti-PD-L1 antibody) is about 300mg to about 900mg of the antibody administered about weekly. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 300mg to about 850mg, about 300mg to about 800mg, about 300mg to about 750mg, about 300mg to about 700mg, about 300mg to about 650mg, about 300mg to about 600mg, about 350mg to about 900mg, about 350mg to about 850mg, about 350mg to about 800mg, about 350mg to about 750mg, about 350mg to about 700mg, about 350mg to about 650mg, about 350mg to about 600mg, about 400mg to about 900mg, about 400mg to about 850mg, about 400mg to about 800mg, about 400mg to about 750mg, about 400mg to about 700mg, about 400mg to about 650mg, about 400mg to about 600mg, about 450 to about 900mg, about 450 to about 850mg, about 450 to about 800mg, about 450mg to about 750mg about 450mg to about 700mg, about 450mg to about 650mg, about 450mg to about 600mg, about 500mg to about 900mg, about 500mg to about 850mg, about 500mg to about 800mg, about 500mg to about 700mg, about 500mg to about 650mg, about 500mg to about 600mg, about 550mg to about 900mg, about 550mg to about 850mg, about 550mg to about 800mg, about 550mg to about 750mg, about 550mg to about 700mg, about 550mg to about 650mg, about 550mg to about 600mg, about 600mg to about 900mg, about 600mg to about 850mg, about 600mg to about 800mg, about 600mg to about 750mg, about 600mg to about 700mg, about 600mg to about 650mg, about 575mg to about 625mg, about 575mg to about 600mg, or about 600mg to about 625 mg. In some aspects, the dosage of the antibody (e.g., the anti-PD-L1 antibody) is about 400mg to about 800mg of the antibody administered about weekly. In some aspects, the dosage of the antibody (e.g., the anti-PD-L1 antibody) is about 500mg to about 700mg of the antibody administered about weekly. In some aspects, the dosage of the antibody (e.g., the anti-PD-L1 antibody) is about 550mg to about 650mg of the antibody administered about weekly. In some aspects, the dosage of the antibody (e.g., the anti-PD-L1 antibody) is about 575mg to about 625mg of the antibody administered per week.

In some aspects, the antibody is administered at a dose of about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 730mg, about 740mg, about 750mg, about 760mg, about 770mg, about 780mg, about 790mg, about 800mg, about 810mg, about 820mg, about 830mg, about 840mg, about 850mg, about 860mg, about 870mg, about 880mg, about 890mg, or about 900mg per week. In some aspects, the antibody is administered at a dose of about 500mg about weekly. In some aspects, the antibody is administered at a dose of about 550mg per week. In some aspects, the antibody is administered at a dose of about 575mg per week. In some aspects, the antibody is administered at a dose of about 600mg about weekly. In some aspects, the antibody is administered at a dose of about 625mg per week. In some aspects, the antibody is administered at a dose of about 650mg per week. In some aspects, the antibody is administered at a dose of about 700mg about weekly.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600 mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600mg in a total administered volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 600mg in a total administration volume of about 5 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 600mg in a total administration volume of about 5 mL.

In some aspects, a dose of about 600mg of the antibody comprises three subcutaneous unit doses. In some aspects, at least one of the three subcutaneous unit doses comprises about 200mg of the antibody. In some aspects, each of the three subcutaneous unit doses comprises about 200mg of the antibody. In some aspects, at least one of the three subcutaneous unit doses comprises about 200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 200mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the three subcutaneous unit doses comprises about 200mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the three subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site and the third subcutaneous unit dose is administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, and the third subcutaneous unit dose is administered at a third body site.

In some aspects, a dose of about 600mg of the antibody comprises at least four subcutaneous unit doses. In some aspects, at least one of the four subcutaneous unit doses comprises about 150mg of the antibody. In some aspects, at least one of the four subcutaneous unit doses comprises about 150mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 150mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the four subcutaneous unit doses comprises greater than about 150mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the four subcutaneous unit doses comprises about 150mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the four subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site, and the third subcutaneous unit dose and the fourth subcutaneous unit dose are administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, the third subcutaneous unit dose is administered at a third body site, and the fourth subcutaneous unit dose is administered at a fourth body site.

In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 900mg to about 1800mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered in a dose of about 900mg to about 1750mg, about 900mg to about 1700mg, about 900mg to about 1650mg, about 900mg to about 1600mg, about 900mg to about 1550mg, about 900mg to about 1500mg, about 900mg to about 1450mg, about 900mg to about 1400mg, about 900mg to about 1350mg, about 900mg to about 1300mg, about 900mg to about 1250mg, about 900mg to about 1200mg, about 950mg to about 1500mg, about 950mg to about 1450mg, about 950mg to about 1400mg, about 950mg to about 1350mg, about 950mg to about 1300mg, about 950mg to about 1250mg, about 950mg to about 1200mg, about 1000mg to about 1500mg, about 1000mg to about 1450mg, about 1000mg to about 1400mg, about 1000mg to about 1350mg, about 1000mg to about 1250mg, about 1000mg to about 1200mg, about 1050mg to about 1050mg, about 1050mg to about 1500mg, about 1050 mg. About 1050 to about 1400mg, about 1050 to about 1350mg, about 1050 to about 1300mg, about 1050 to about 1250mg, about 1050 to about 1200mg, about 1100 to about 1500mg, about 1100 to about 1450mg, about 1100 to about 1400mg, about 1100 to about 1350mg, about 1100 to about 1300mg, about 1100 to about 1250mg, about 1100 to about 1200mg, about 1150 to about 1500mg, about 1150 to about 1450mg, about 1150 to about 1400mg, about 1150 to about 1350mg, about 1150 to about 1300mg, about 1150 to about 1250mg, about 1150 to about 1200mg, about 1200 to about 1500mg, about 1200 to about 1450mg, about 1200 to about 1400mg, about 1200 to about 1350mg, about 1200 to about 1250mg, about 1175 to about 1225mg, about 1175 to about 1200mg, or about 1200 to about 1225mg of the antibody. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 1000mg to about 1400mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 1100mg to about 1300mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 1150mg to about 1250mg of the antibody every two weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 1175mg to about 1225mg of the antibody every two weeks.

In some aspects, the antibody is administered at a dose of about 900mg, about 950mg, about 1000mg, about 1010mg, about 1020mg, about 1030mg, about 1040mg, about 1050mg, about 1060mg, about 1070mg, about 1080mg, about 1090mg, about 1100mg, about 1110mg, about 1120mg, about 1130mg, about 1140mg, about 1150mg, about 1160mg, about 1170mg, about 1180mg, about 1190mg, about 1200mg, about 1210mg, about 1220mg, about 1230mg, about 1240mg, about 1250mg, about 1260mg, about 1270mg, about 1280mg, about 1290mg, about 1300mg, about 1310mg, about 1320mg, about 1330mg, about 1340mg, about 1350mg, about 1360mg, about 1370mg, about 1380mg, about 1390mg, about 1400mg, about 1410mg, about 1420mg, about 1440mg, about 1450mg, about 1430mg, about 1470mg, about 1490mg, about 148 mg, about 1600mg, about 1500mg, about 1750mg, about 1500mg, or about 1800mg every two weeks. In some aspects, the antibody is administered at a dose of about 1100mg every two weeks. In some aspects, the antibody is administered at a dose of about 1150mg every two weeks. In some aspects, the antibody is administered at a dose of about 1175mg every two weeks. In some aspects, the antibody is administered at a dose of about 1200mg every two weeks. In some aspects, the antibody is administered at a dose of about 1225mg every two weeks. In some aspects, the antibody is administered at a dose of about 1250mg every two weeks. In some aspects, the antibody is administered at a dose of about 1300mg every two weeks.

In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 1200 mg. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of less than about 1200mg in a total administered volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 1200mg in a total administration volume of about 2 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of about 1200mg in a total administration volume of about 5 mL. In some aspects, the dosage of the antibody comprises a single subcutaneous unit dose of greater than about 1200mg in a total administration volume of about 5 mL.

In some aspects, the two, three, four, six, or at least eight subcutaneous unit doses are administered on the same day.

In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 2100mg to about 2700mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 2100mg to about 2650mg, about 2100mg to about 2600mg, about 2100mg to about 2550mg, about 2100mg to about 2500mg, about 2100mg to about 2450mg, about 2100mg to about 2400mg, about 2200mg to about 2700mg, about 2200mg to about 2650mg, about 2200mg to about 2600mg, about 2200mg to about 2550mg, about 2200mg to about 2500mg, about 2200mg to about 2450mg, about 2200mg to about 2400mg, about 2300mg to about 2700mg, about 2300mg to about 2650mg, about 2300mg to about 2600mg, about 2300mg to about 2550mg, about 2300mg to about 2500mg about 2300mg to about 2450mg, about 2300mg to about 2400mg, about 2350mg to about 2500mg, about 2350mg to about 2700mg, about 2350mg to about 2650mg, about 2350mg to about 2600mg, about 2350mg to about 2550mg, about 2350mg to about 2500mg, about 2350mg to about 2450mg, about 2350mg to about 2400mg, about 2400mg to about 2700mg, about 2400mg to about 2650mg, about 2400mg to about 2600mg, about 2400mg to about 2550mg, about 2400mg to about 2500mg, about 2400mg to about 2450mg, about 2400mg to about 2425mg, about 2375mg to about 2425mg, or about 2375mg to about 2400mg of the antibody. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 2200mg to about 2600mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 2300mg to about 2500mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 2350mg to about 2450mg of the antibody every four weeks. In some aspects, the antibody (e.g., the anti-PD-L1 antibody) is administered at a dose of about 2375mg to about 2425mg of the antibody every four weeks.

In some aspects, the antibody is administered at a dose of about 2100mg, about 2150mg, about 2200mg, about 2250mg, about 2300mg, about 2350mg, about 2400mg, about 2450mg, about 2500mg, about 2550mg, about 2600mg, about 2650mg, or about 2700mg every four weeks. In some aspects, the antibody is administered at a dose of about 2300mg every four weeks. In some aspects, the antibody is administered at a dose of about 2350mg every four weeks. In some aspects, the antibody is administered at a dose of about 2400mg every four weeks. In some aspects, the antibody is administered at a dose of about 2450mg about every four weeks. In some aspects, the antibody is administered at a dose of about 2500mg every four weeks.

In some aspects, the dose of the antibody comprises a single subcutaneous dose comprising about 2400mg of the antibody. In some aspects, a single subcutaneous dose of the antibody comprises greater than about 2400mg of the antibody in a total volume of about 2 mL. In some aspects, the single subcutaneous unit dose comprises greater than about 2400mg of the antibody in a total volume of about 5 mL.

In some aspects, the dosage of the antibody comprises two, three, four, six, or at least eight subcutaneous unit doses. In some aspects, the dosage of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 1200mg of the antibody. In some aspects, at least one of the two subcutaneous unit doses comprises about 1200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 1200mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the two subcutaneous unit doses comprises greater than about 1200mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the two subcutaneous unit doses comprises less than about 1200mg of the antibody in a total volume of less than about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In certain aspects, at least one of the two subcutaneous unit doses comprises less than about 1200mg of the antibody in a total volume of about 2 mL. In some aspects, the two subcutaneous unit doses are administered to a single body location of the subject. In some aspects, the two subcutaneous unit doses are administered to two different body locations of the subject.

In some aspects, a dose of about 2400mg of the antibody comprises three subcutaneous unit doses. In some aspects, at least one of the three subcutaneous unit doses comprises about 800mg of the antibody. In some aspects, each of the three subcutaneous unit doses comprises about 800mg of the antibody. In some aspects, at least one of the three subcutaneous unit doses comprises about 800mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 800mg of the antibody in a total volume of about 2 mL. In some aspects, at least one of the three subcutaneous unit doses comprises greater than about 800mg of the antibody in a total volume of about 5 mL. In some aspects, at least one of the three subcutaneous unit doses comprises about 800mg of the antibody in a total volume of about 5mL (e.g., less than about 4.5mL, less than about 4.0mL, less than about 3.5mL, less than about 3.0mL, less than about 2.5mL, or less than about 2.0 mL). In some aspects, at least two of the three subcutaneous unit doses are administered to at least two different body locations of the subject. In some aspects, the first subcutaneous unit dose and the second subcutaneous unit dose are administered at a first body site and the third subcutaneous unit dose is administered at a second body site. In some aspects, the first subcutaneous unit dose is administered at a first body site, the second subcutaneous unit dose is administered at a second body site, and the third subcutaneous unit dose is administered at a third body site.

In some aspects, the anti-PD-L1 antibody comprises alemtuzumab, which is administered subcutaneously, about once per week, about once every two weeks, about once every three weeks, or about once every four weeks. In some aspects, about 1000mg to about 1400mg of alemtuzumab is administered subcutaneously, about once every two weeks. In some aspects, about 1100mg, about 1110mg, about 1120mg, about 1130mg, about 1140mg, about 1150mg, about 1160mg, about 1170mg, about 1180mg, about 1190mg, about 1200mg, about 1210mg, about 1220mg, about 1230mg, about 1240mg, about 1250mg, about 1260mg, about 1270mg, about 1280mg, about 1290mg, or about 1300mg of alemtuzumab is administered subcutaneously, about once every two weeks. In some aspects, at least about 1200mg of alemtuzumab is administered subcutaneously, about once every two weeks. In some aspects, about 1700mg to about 1900mg of alemtuzumab is administered subcutaneously, about once every three weeks. In some aspects, about 1700mg, about 1710mg, about 1720mg, about 1730mg, about 1740mg, about 1750mg, about 1760mg, about 1770mg, about 1780mg, about 1790mg, about 1800mg, about 1810mg, about 1820mg, about 1830mg, about 1840mg, about 1850mg, about 1860mg, about 1870mg, about 1880mg, about 1890mg, or about 1900mg of alemtuzumab is administered subcutaneously, about once every three weeks. In some aspects, at least about 1800mg of alemtuzumab is administered subcutaneously, about once every three weeks. In some aspects, the dose of alemtuzumab is administered in a volume of at least about 2mL to at least about 20 mL.

II.A.3 endoglycosidase hydrolase administration

In some aspects, the pharmaceutical composition comprises an endoglycosidase hydrolase. Any endoglycosidase hydrolase may be used in the pharmaceutical compositions disclosed herein. In some aspects, the endoglycosidase hydrolase cleaves hyaluronic acid at a hexosamine β (1-4) or (1-3) junction. In some aspects, the endoglycosidase hydrolase comprises the catalytic domain of hyaluronidase PH-20 (HuPH 20), HYAL1, HYAL2, HYAL3, HYAL4, or HYALPS 1.

In some aspects, the endoglycosidase hydrolase comprises a hyaluronidase. In some aspects, the endoglycosidase hydrolase comprises a hyaluronidase selected from the group consisting of HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, any variant thereof, and any isoform. In some aspects, the endoglycosidase hydrolase comprises rHuPH20 or a fragment thereof. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to amino acids 36-490 of SEQ ID No. 1. In some aspects, the endoglycosidase hydrolase comprises the catalytic domain of rHuPH20 (UniProt ID No. P38567-1). In some aspects, the endoglycosidase hydrolase comprises the rHuPH20 mature peptide (amino acids 36-490 of SEQ ID NO: 1A).

Table 1A: amino acid sequence of rHuPH20

Signal peptide: underline; mature protein: bold; pro peptide: italics.

In some aspects, the subcutaneous injection is Halozyme Therapeutics-basedDrug delivery techniques (see U.S. Pat. No. 7,767,429, which is incorporated herein by reference in its entirety). />The use of co-formulations of antibodies with recombinant human hyaluronidase (rHuPH 20) eliminates the traditional limitation of the volume of biologicals and drugs that can be delivered subcutaneously due to the extracellular matrix (see U.S. Pat. No. 7,767,429). In some aspects, the pharmaceutical compositions used in the present disclosure may further comprise a recombinant human hyaluronidase (e.g., rHuPH 20).

Recombinant human hyaluronidase PH20 (rHuPH 20, halozyme Therapeutics inc.) is a glycosylated 447 amino acid single chain recombinant human polypeptide that locally depolymerizes hyaluronic acid in the Subcutaneous (SC) gap at the injection site. Hyaluronic acid is a repeating polymer of N-acetyl-glucosamine and glucuronic acid that contributes to the soluble gel-like components of the extracellular matrix of the skin. Acidolysis of hyaluronic acid by rHuPH20 resulted in a short decrease in viscosity of the gel-like phase of the extracellular matrix and increased water conductivity, thereby promoting dispersion and absorption of the injected drug (see rHuPH20 IB). The use of rHuPH20 enables delivery of large volumes for rapid SC injection (e.g., about 2mL to 20 mL), which can shorten the dosing time, reduce the frequency of administration, and enable potential improvements to PK profile of co-administered drugs, including improved absorption, increased bioavailability, faster time to maximum concentration (Tmax), increased maximum concentration (Cmax), and reduced PK variability.

rHuPH20 had a half-life of <30 minutes in skin and the local permeation barrier in these tissues was restored to pre-injection levels within 24 to 48 hours after hyaluronidase injection. Studies have shown that rHuPH20 is not systemically detectable in healthy volunteers and patients following SC administration at doses of 10,000u and 30,000U. Another study of the PK of rHuPH20 (Halozyme study HALO-104-104) demonstrated that the plasma concentration of rHuPH20 decreased rapidly, t1/2 was extremely short (. Ltoreq.10.4 min), and that the plasma concentration became undetectable (< 0.03 ng/mL) within 1.5 hours after the end of IV infusion at an IV dose of 10,000 or 30,000 units of rHuPH 20.

Among healthy participants, pediatric participants who are dehydrated, participants who are in the end care and palliative care, participants who have type 1 and type 2 diabetes, and participants who have rheumatoid arthritis, subcutaneous injections of rHuPH20 are generally well tolerated. Subcutaneous injections of rHuPH20 alone or co-administered with lactate ringer solution, physiological saline, co-injected drugs (morphine, ceftriaxone, insulin and insulin analogues) or biologic products (immunoglobulin G [ IgG ] and adalimumab) are well tolerated.

In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the alpha helical region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the linker region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof. In some aspects, the endoglycosidase hydrolase comprises a modified hyaluronidase in which one or more N-terminal and/or C-terminal amino acids are deleted relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

In some aspects, the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises one or more amino acid substitutions in the alpha helical region, the linker region, or both the alpha helical region and the linker region relative to wild-type rHuPH 20. In some aspects, the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises a deletion of one or more N-terminal amino acids, one or more C-terminal amino acids, or one or more N-terminal amino acids and one or more C-terminal amino acids relative to wild-type rHuPH 20. In some aspects, the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises one or more amino acid substitutions in the alpha helical region, the linker region, or both the alpha helical region and the linker region relative to wild-type rHuPH 20; and wherein the modified rHuPH20 comprises a deletion of one or more N-terminal amino acids, one or more C-terminal amino acids, or one or more N-terminal amino acids and one or more C-terminal amino acids relative to wild-type rHuPH 20.

Further non-limiting examples of endoglycosidase hydrolases are found in EP3636752, which is incorporated herein by reference in its entirety.

In some aspects, the endoglycosidase hydrolase is any polypeptide having endoglycosidase hydrolase activity disclosed in the following documents: U.S. Pat. No. 3,182; US 10,865,400; US 11,041,149; US 11,066,656; US 8,927,249; US 9,284,543; US 10,588,983; US 10/328,130 and/or US 9,993,529, each of which is incorporated herein by reference in its entirety. In some aspects, the endoglycosidase hydrolase is any polypeptide having endoglycosidase hydrolase activity disclosed in the following documents: international publication Nos. WO/13/102144, WO/10/077297, WO/15/003167, WO/04/078140, WO/09/128917, WO/12/174478 and/or WO/12/174480, each of which is incorporated herein by reference in its entirety. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs 5-52. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs 5-52.

In some aspects, the endoglycosidase hydrolase is any polypeptide having endoglycosidase hydrolase activity disclosed in the following documents: U.S. patent application publication nos. US 2021155913 A1 and/or US 2021363270 A1; and/or International publication Nos. WO/20/022791, WO/20/197230 and/or WO/21/150079; each of which is incorporated herein by reference in its entirety. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs 53-263. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID No. 92. In some aspects, the endoglycosidase hydrolase comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs 53-263. In some aspects, the endoglycosidase hydrolase comprises the amino acid sequence set forth in SEQ ID NO. 92. In some aspects, the endoglycosidase hydrolase is HP46 (SEQ ID NO:44 of International publication No. WO/20/197230).

In certain aspects, the pharmaceutical compositions disclosed herein comprise hyaluronidase. In some aspects, the pharmaceutical composition comprises a hyaluronidase in a concentration sufficient for administration of at least about 20,000 units of the hyaluronidase. In some aspects, the hyaluronidase is rHuPH20. In other aspects, the pharmaceutical composition does not comprise hyaluronidase.

In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the endoglycosidase hydrolase (e.g., rHuPH 20) are administered together. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the endoglycosidase hydrolase (e.g., rHuPH 20) are administered in parallel. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the endoglycosidase hydrolase (e.g., rHuPH 20) are administered sequentially.

In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the endoglycosidase hydrolase (e.g., rHuPH 20) are present in a single pharmaceutical composition.

In some aspects, the dose comprises at least about 5000 units to at least about 100,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dosage comprises at least about 5000 units, at least about 10,000 units, at least about 15,000 units, at least about 20,000 units, at least about 25,000 units, at least about 30,000 units, at least about 35,000 units, at least about 40,000 units, at least about 45,000 units, at least about 50,000 units, at least about 55,000 units, at least about 60,000 units, at least about 65,000 units, at least about 70,000 units, at least about 75,000 units, at least about 80,000 units, at least about 85,000 units, at least about 90,000 units, at least about 95,000 units, or at least about 100,000 units of endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 20,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 30,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 40,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 50,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 60,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 70,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 80,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 90,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the dose comprises at least about 100,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20).

Those skilled in the art will readily appreciate that the amount of the endoglycosidase hydrolase (e.g., rHuPH 20) may be expressed in units or the amount of the endoglycosidase hydrolase (e.g., rHuPH 20) may be expressed in mg (or in other weight-based units). For example, in some aspects, the dose comprises an endoglycosidase hydrolase (e.g., rHuPH 20) in an amount expressed as at least about 500U or at least about 0.00455 mg. In another example, in some aspects, the dose comprises an endoglycosidase hydrolase (e.g., rHuPH 20) in an amount expressed as at least about 2000U or at least about 0.0182 mg.

anti-PD-1 antibodies useful in the present disclosure

anti-PD-1 antibodies known in the art may be used in the presently described compositions and methods. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity have been disclosed in U.S. patent No. 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. patent No. 8,008,449 have been demonstrated to exhibit one or more of the following characteristics: (a) At 1x10 ^-7 M or less K _D Binding to human PD-1 as determined by surface plasmon resonance using a Biacore biosensor system; (b) does not substantially bind to human CD28, CTLA-4 or ICOS; (c) Increasing T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (d) increasing interferon-gamma production in the MLR assay; (e) increasing IL-2 secretion in the MLR assay; (f) binds to human PD-1 and cynomolgus PD-1; (g) inhibiting the binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulating an antigen-specific memory response; (i) stimulating an antibody response; and (j) inhibiting tumor cell growth in vivo. anti-PD-1 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one (and in some aspects at least five) of the foregoing characteristics.

Other anti-PD-1 monoclonal antibodies have been described, for example, in the following: us patent numbers 6,808,710, 7,488,802, 8,168,757 and 8,354,509, us publication numbers 2016/0272708, and PCT publication numbers WO 2012/145493, WO 2008/156712, WO 2015/112900, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO2014/179664, WO 2017/020291, WO 2017/020858, WO 2016/197367, WO 2017/0245515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/1327, WO 2017/024665, WO 2017/025016, WO 2017/10646, WO 2017/0202465, WO 2017/025016, WO 2017/1335, WO 2017/19846, WO 061/02465, WO 2017/133540 are each incorporated by reference in their entirety.

In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as5C4, BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (Merck; also called +.>Lanbulizumab and MK-3475; see WO 2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cimipne Li Shan anti (Regeneron; also known as REGN-2810; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as terep Li Shan antibody; see Si-Yang Liu et al, J.Hematol. Oncol.10:136 (2017)), BGB-A317 (Beigene; also known as tirelizumab; see WO 2015/35606 and US 2015/0079209), incsshr 1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; si-Yang Liu et al, J.Hematol. Oncol.10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014/179664), GLS-010 (Wuxi/Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al, J.Hematol. Oncol.10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (agalus; see WO 2017/040790), MGA012 (macrogenetics, see WO 2017/19846), BCD-100 (Biocad; kaplon et al, mAbs 10 (2): 183-203 (2018)), IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825 and WO 2017/133540); and Sashan Li Shan resistance (PF-06801591).

In one aspect, the anti-PD-1 antibody is nivolumab. Nivolumab is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively blocks interactions with PD-1 ligands (PD-L1 and PD-L2), thereby blocking down-regulation of anti-tumor T cell function (U.S. Pat. No. 8,008,449; wang et al, 2014Cancer Immunol Res.2 (9): 846-56). The heavy and light chain variable regions of nivolumab are shown in Table 1B (SEQ ID NOS: 2 and 3). In some aspects, the anti-PD-1 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 2 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 3. In some aspects, the antibodies comprise heavy chain Complementarity Determining Region (CDR) 1, CDR2 and CDR3 sequences comprising the amino acid sequences of heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO. 2. In some aspects, the antibodies comprise light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of light chain CDR1, CDR2 and CDR3 of SEQ ID NO 3.

Table 1B: anti-PD-1 antibody sequences

In another aspect, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against human cell surface receptor PD-1 (apoptosis protein-1 or apoptosis protein-1). Pembrolizumab is described, for example, in U.S. patent nos. 8,354,509 and 8,900,587.

anti-PD-1 antibodies useful in the disclosed compositions and methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein (e.g., nivolumab) for binding to human PD-1 (see, e.g., U.S. patent nos. 8,008,449 and 8,779,105;WO 2013/173223). In some aspects, the anti-PD-1 antibodies bind to the same epitope as any of the anti-PD-1 antibodies described herein (e.g., nivolumab). The ability of antibodies to cross-compete for binding to an antigen suggests that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have very similar functional properties to the reference antibody (e.g., nivolumab) due to their binding to the same epitope region of PD-1. Standard PD-1 binding assays (such as Biacore assays, ELISA assays, or flow cytometry) can be readily identified based on their ability to cross-compete with nivolumab (see, e.g., WO 2013/173223).

In certain aspects, the antibody that cross-competes with nivolumab for binding to human PD-1 or binds to the same epitope region of human PD-1 antibody as nivolumab is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are chimeric, engineered or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies may be prepared and isolated by methods well known in the art.

anti-PD-1 antibodies useful in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the antibodies described above. It is well documented that the antigen binding function of an antibody can be performed by fragments of a full length antibody.

anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits similar functional properties as an intact antibody in terms of inhibiting ligand binding and up-regulating the immune system. In certain aspects, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1.

In some aspects, the anti-PD-1 antibody is administered once every 2, 3, 4, 5, 6, 7, or 8 weeks at a dose ranging from 0.1mg/kg to 20.0mg/kg body weight (e.g., once every 2, 3, or 4 weeks of 0.1mg/kg to 10.0mg/kg body weight). In other aspects, the anti-PD-1 antibody is administered at a dose of about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, or 10mg/kg body weight once every 2 weeks. In other aspects, the anti-PD-1 antibody is administered at a dose of about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, or 10mg/kg body weight once every 3 weeks. In one aspect, the anti-PD-1 antibody is administered at a dose of about 5mg/kg body weight about once every 3 weeks. In another aspect, the anti-PD-1 antibody (e.g., nivolumab) is administered at a dose of about 3mg/kg body weight about once every 2 weeks. In other aspects, the anti-PD-1 antibody (e.g., pembrolizumab) is administered at a dose of about 2mg/kg body weight about once every 3 weeks.

anti-PD-1 antibodies useful in the present disclosure may be administered as flat doses. In some aspects, the anti-PD-1 antibody is administered at a flat dose as follows: about 100 to about 1000mg, about 100 to about 900mg, about 100 to about 800mg, about 100 to about 700mg, about 100 to about 600mg, about 100 to about 500mg, about 200 to about 1000mg, about 200 to about 900mg, about 200 to about 800mg, about 200 to about 700mg, about 200 to about 600mg, about 200 to about 500mg, about 200 to about 480mg, or about 240 to about 480mg. In one aspect, the anti-PD-1 antibody is administered as a flat dose at dosing intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks as follows: at least about 200mg, at least about 220mg, at least about 240mg, at least about 260mg, at least about 280mg, at least about 300mg, at least about 320mg, at least about 340mg, at least about 360mg, at least about 380mg, at least about 400mg, at least about 420mg, at least about 440mg, at least about 460mg, at least about 480mg, at least about 500mg, at least about 520mg, at least about 540mg, at least about 550mg, at least about 560mg, at least about 580mg, at least about 600mg, at least about 620mg, at least about 640mg, at least about 660mg, at least about 680mg, at least about 700mg, or at least about 720mg. In another aspect, the anti-PD-1 antibody is administered as a flat dose at dosing intervals of about 1, 2, 3, or 4 weeks as follows: about 200mg to about 800mg, about 200mg to about 700mg, about 200mg to about 600mg, about 200mg to about 500mg.

In some aspects, the anti-PD-1 antibody is administered as a flat dose of about 200mg, about once every 3 weeks. In other aspects, the anti-PD-1 antibody is administered as a flat dose of about 200mg, about once every 2 weeks. In other aspects, the anti-PD-1 antibody is administered as a flat dose of about 240mg, about once every 2 weeks. In certain aspects, the anti-PD-1 antibody is administered as a flat dose of about 480mg, about once every 4 weeks.

In some aspects, the nivolumab is administered at a flat dose of about 240mg about every 2 weeks. In some aspects, the nivolumab is administered at a flat dose of about 240mg about once every 3 weeks. In some aspects, the nivolumab is administered at a flat dose of about 360mg about once every 3 weeks. In some aspects, the nivolumab is administered at a flat dose of about 480mg about once every 4 weeks. In some aspects, the nivolumab is administered at a flat dose of about 720mg about once every 6 weeks. In some aspects, the nivolumab is administered at a flat dose of about 960mg about once every 8 weeks.

In some aspects, pembrolizumab is administered at a flat dose of about 200mg about once every 2 weeks. In some aspects, pembrolizumab is administered at a flat dose of about 200mg about once every 3 weeks. In some aspects, pembrolizumab is administered at a flat dose of about 400mg about once every 4 weeks.

In some aspects, the pharmaceutical composition comprises a bispecific or multispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises an anti-PD-1 antigen-binding portion (e.g., scFv of nivolumab). In some aspects, the second antigen binding portion is an antigen binding portion of any one of the antibodies disclosed herein. In some aspects, the second antigen binding portion is an antigen binding portion of an anti-LAG-3 antibody (e.g., a rayleigh Li Shan antibody).

In some aspects, the pharmaceutical composition comprises a multispecific antibody comprising a first antigen-binding portion, a second antigen-binding portion, and at least a third antigen-binding portion, wherein the first antigen-binding portion comprises an anti-PD-1 antigen-binding portion (e.g., scFv of nivolumab).

II.C. anti-PD-L1 antibodies useful in the present disclosure

In certain aspects, in any of the methods disclosed herein, the anti-PD-1 antibody is substituted with an anti-PD-L1 antibody. Any anti-PD-L1 antibody can be used in the compositions and methods of the present disclosure. Examples of anti-PD-L1 antibodies that can be used in the compositions and methods of the present disclosure include the antibodies disclosed in U.S. patent No. 9,580,507. The anti-PD-L1 human monoclonal antibodies disclosed in us patent No. 9,580,507 have been demonstrated to exhibit one or more of the following characteristics: (a) At 1x10 ^-7 M or less K _D Binding to human PD-L1, e.g. using BiThe acore biosensor system was determined by surface plasmon resonance; (b) Increasing T cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (c) increasing interferon-gamma production in the MLR assay; (d) increasing IL-2 secretion in an MLR assay; (e) stimulating an antibody response; and (f) reversing the effect of the T regulatory cells on T cell effector cells and/or dendritic cells. anti-PD-L1 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one (and in some aspects at least five) of the foregoing characteristics.

In certain aspects, the anti-PD-L1 antibody is selected from BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO 2013/173223), alemtuzumab (Roche; also known asMPDL3280A, RG7446; see US 8,217,149; see also Herbst et al (2013) J Clin Oncol 31 (journal): 3000), cerstuzumab (AstraZeneca; also known as IMFINZI ^TM MEDI-4736; see WO 2011/066389), avermectin (Pfizer; also called +.>MSB-0010718C; see WO 2013/079174), STI-1014 (Sorrento; see WO 2013/181634), CX-072 (Cytomx; see WO 2016/14991), KN035 (3D Med/Alphamab; see Zhang et al, cell discovery.7:3 (3 months of 2017)), LY3300054 (Eli Lilly co.; see, e.g., WO 2017/034916), BGB-a333 (BeiGene; see Desai et al, JCO 36 (15 journal): TPS3113 (2018)), CK-301 (Checkpoint Therapeutics; see Gorelik et al, AACR: abstract 4606 (month 4 of 2016)).

In certain aspects, the PD-L1 antibody is alemtuzumabAlemtuzumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

In certain aspects, the PD-L1 antibody is dimaruzumab (IMFINZI ^TM ). Devaluzumab is a humanIgG1 kappa monoclonal anti-PD-L1 antibody.

In certain aspects, the PD-L1 antibody is avermectinAvermectin is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

anti-PD-L1 antibodies useful in the disclosed compositions and methods also include isolated antibodies that specifically bind to human PD-L1 and cross-compete with any anti-PD-L1 antibody disclosed herein (e.g., alemtuzumab, dimaruzumab, and/or avistuzumab) for binding to human PD-L1. In some aspects, the anti-PD-L1 antibody binds to the same epitope as any anti-PD-L1 antibody described herein (e.g., alemtuzumab, dimaruzumab, and/or avistuzumab). The ability of antibodies to cross-compete for binding to an antigen suggests that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have very similar functional properties as the reference antibodies (e.g., alemtuzumab and/or avistuzumab) due to their binding to the same epitope region of PD-L1. Standard PD-L1 binding assays (such as Biacore assays, ELISA assays, or flow cytometry) can be readily identified based on their ability to cross-compete with the cross-competing antibodies with the alemtuzumab and/or avistuzumab (see, e.g., WO 2013/173223).

In certain aspects, antibodies cross-competing with the alemtuzumab, the divaruzumab, and/or the avermectin bind to human PD-L1 or the same epitope region as the alemtuzumab, the divaruzumab, and/or the avermectin binds to human PD-L1 antibody are monoclonal antibodies. For administration to a human subject, these cross-competing antibodies are chimeric, engineered or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies may be prepared and isolated by methods well known in the art.

anti-PD-L1 antibodies useful in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the antibodies described above. It is well documented that the antigen binding function of an antibody can be performed by fragments of a full length antibody.

anti-PD-L1 antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-L1 with high specificity and affinity, block binding of PD-1, and inhibit immunosuppression of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits similar functional properties as an intact antibody in terms of inhibiting receptor binding and up-regulating the immune system. In certain aspects, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with alemtuzumab, dimaruzumab, and/or avistuzumab for binding to human PD-L1.

The anti-PD-L1 antibodies useful in the present disclosure may be any PD-L1 antibody that specifically binds to PD-L1, such as an antibody that cross-competes with dimvaluzumab, avistuzumab, or alemtuzumab for binding to human PD-1, such as an antibody that binds to the same epitope as dimvaluzumab, avistuzumab, or alemtuzumab. In a particular aspect, the anti-PD-L1 antibody is cerulomumab. In other aspects, the anti-PD-L1 antibody is avilamab. In some aspects, the anti-PD-L1 antibody is alemtuzumab.

In some aspects, the anti-PD-L1 antibody is administered at a dose ranging from: about 0.1mg/kg to about 20.0mg/kg of body weight, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg or about 20mg/kg, about once every 2, 3, 4, 5, 6, 7 or 8 weeks.

In some aspects, the anti-PD-L1 antibody is administered at a dose of about 15mg/kg body weight, about once every 3 weeks. In other aspects, the anti-PD-L1 antibody is administered at a dose of about 10mg/kg body weight, about once every 2 weeks.

In other aspects, anti-PD-L1 antibodies useful in the present disclosure are flat doses. In some aspects, the anti-PD-L1 antibody is administered as a flat dose of: about 200mg to about 1600mg, about 200mg to about 1500mg, about 200mg to about 1400mg, about 200mg to about 1300mg, about 200mg to about 1200mg, about 200mg to about 1100mg, about 200mg to about 1000mg, about 200mg to about 900mg, about 200mg to about 800mg, about 200mg to about 700mg, about 200mg to about 600mg, about 700mg to about 1300mg, about 800mg to about 1200mg, about 700mg to about 900mg, or about 1100mg to about 1300mg. In some aspects, the anti-PD-L1 antibody is administered as a flat dose at dosing intervals of about 1, 2, 3, or 4 weeks as follows: at least about 240mg, at least about 300mg, at least about 320mg, at least about 400mg, at least about 480mg, at least about 500mg, at least about 560mg, at least about 600mg, at least about 640mg, at least about 700mg, at least 720mg, at least about 800mg, at least about 840mg, at least about 880mg, at least about 900mg, at least 960mg, at least about 1000mg, at least about 1040mg, at least about 1100mg, at least about 1120mg, at least about 1200mg, at least about 1280mg, at least about 1300mg, at least about 1360mg, or at least about 1400mg. In some aspects, the anti-PD-L1 antibody is administered as a flat dose of about 1200mg, about once every 3 weeks. In other aspects, the anti-PD-L1 antibody is administered as a flat dose of about 800mg, about once every 2 weeks. In other aspects, the anti-PD-L1 antibody is administered as a flat dose of about 840mg, about once every 2 weeks.

In some aspects, the alemtuzumab is administered as a flat dose of about 1200mg, about once every 3 weeks. In some aspects, the alemtuzumab is administered as a flat dose of about 800mg, about once every 2 weeks. In some aspects, the alemtuzumab is administered as a flat dose of about 840mg, about once every 2 weeks.

In some aspects, the avermectin is administered as a flat dose of about 800mg, about once every 2 weeks.

In some aspects, the divaruzumab is administered at a dose of about 10mg/kg, about once every 2 weeks. In some aspects, the divaruzumab is administered as a flat dose of about 800mg/kg, about once every 2 weeks. In some aspects, the divaruzumab is administered as a flat dose of about 1200mg/kg, about once every 3 weeks.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) PD-L1 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) PD-L1 and (ii) CD3.

II.D. combination therapy

In some aspects of the disclosure, the methods disclosed herein comprise administering an anti-PD-1 antibody (or an anti-PD-L1 antibody) in combination with an additional anti-cancer therapy. The additional anti-cancer therapies may include any therapy for treating a tumor in a subject and/or any standard of care therapy, as disclosed herein. In some aspects, the additional anti-cancer therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some aspects, the additional anti-cancer therapy comprises chemotherapy, including any of the chemotherapy disclosed herein. In some aspects, the additional anti-cancer therapy comprises immunotherapy. In some aspects, the additional anti-cancer therapies comprise administering an antibody or antigen-binding portion thereof that specifically binds CTLA-4, LAG-3, TIGIT, TIM3, NKG2a, OX40, ICOS, MICA, CD137, KIR, tgfβ, IL-10, IL-8, B7-H4, fas ligand, CXCR4, mesothelin, CD27, GITR, or any combination thereof. In some aspects, the additional anti-cancer therapy comprises administration of IL-2 (e.g., modified IL-2, e.g., pegylated IL-2, e.g., bei Peia interleukin). In some aspects, the second therapeutic agent, the third therapeutic agent, or both comprise IL12-Fc (e.g., BMS-986415).

In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody is administered subcutaneously according to any of the methods disclosed herein, and the additional anti-cancer therapy is administered by any suitable route known in the art. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody is administered subcutaneously according to any of the methods disclosed herein, and the additional anti-cancer therapy is administered subcutaneously. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody is administered subcutaneously according to any of the methods disclosed herein, and the additional anti-cancer therapy is administered intravenously. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the additional anti-cancer therapy are administered concurrently. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the additional anti-cancer therapy are administered sequentially. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the additional anti-cancer therapy are administered on the same day. In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the additional anti-cancer therapy are administered at different days.

In some aspects, the anti-PD-1 antibody or the anti-PD-L1 antibody and the additional anti-cancer therapy (e.g., checkpoint inhibitor) are combined in a single formulation.

In some aspects, the methods comprise administering a therapeutically effective amount of an anti-PD-1 antibody and an anti-CTLA-4 antibody, e.g., ipilimumab. In other aspects, the methods comprise administering a therapeutically effective amount of a composition comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody. Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity have been disclosed in U.S. patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described, for example, in the following: U.S. Pat. nos. 5,977,318, 6,051,227, 6,682,736 and 7,034,121, and international publication nos. WO 2012/12244, WO 2007/113648, WO 2016/196237 and WO 2000/037504, each of which is incorporated herein by reference in its entirety. In certain aspects, the CTLA-4 antibody is selected from ipilimumab (also known asMDX-010, 10D1; see U.S. patent No. 6,984,720), MK-1308 (Merck), AGEN-1884 (agalus inc; see WO 2016/196237) and trimethoprim (AstraZeneca; also known as tiximumab, CP-675,206; see WO 2000/037504 and Ribas, update Cancer Ther.2 (3): 133-39 (2007)). In a particular aspect, the anti-CTLA-4 antibody is ipilimumab. In a particular aspect, the CTLA-4 antibody is tremelimumab. In a particular aspect, the CTLA-4 antibody is MK-1308. In a particular aspect, the CTLA-4 antibody is AGEN-1884.

In some aspects, the methods comprise administering a therapeutically effective amount of an anti-PD-1 antibody and an anti-LAG-3 antibody. In other aspects, the methods comprise administering a therapeutically effective amount of a single formulation comprising an anti-PD-1 antibody and an anti-LAG-3 antibody (e.g., a rayleigh Li Shan antibody). In some aspects, the anti-LAG-3 antibody is a rally Li Shan antibody, e.g., BMS-986016 as described in PCT/US13/48999, the teachings of which are hereby incorporated by reference. In some aspects, the anti-LAG-3 antibody cross-competes with rale Li Shan for binding to human LAG-3. In some aspects, the anti-LAG-3 antibody binds to the same epitope as the rale Li Shan antibody. In some aspects, the anti-LAG-3 antibody is a biological analog of the rapa Li Shan antibody. In some aspects, the anti-LAG-3 antibody is LAG-525, MK-4280, REGN3767, TSR-033, TSR-075, sym022, FS-118, or any combination thereof.

In some aspects, the methods comprise administering a therapeutically effective amount of an anti-PD-1 antibody or an anti-PD-L1 antibody according to any of the methods and chemotherapies disclosed herein. In some aspects, the chemotherapy comprises platinum-based therapy. In some aspects, the platinum-based therapy comprises a platinum-based antineoplastic agent selected from the group consisting of: cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate (phenanthriplatin), picoplatin, satraplatin, and any combination thereof. In certain aspects, the platinum-based therapy comprises cisplatin. In a particular aspect, the platinum-based therapy comprises carboplatin. In some aspects, the chemotherapy comprises an anticancer agent selected from the group consisting of: platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab) Erlotinib>Crizotinib->CetuximabAnd any combination thereof. In certain aspects, the chemotherapy comprises platinum-based dual-drug chemotherapy.

II.E. tumors

Certain aspects of the present disclosure relate to methods of treating a subject in need thereof, the methods comprising subcutaneously delivering an anti-PD-1 antibody or an anti-PD-L1 antibody. In some aspects, the subject has cancer (e.g., a tumor derived from cancer). In some aspects, the tumor is derived from a cancer selected from the group consisting of: squamous cell carcinoma (squamous cell carcinoma), small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (renal cancer), clear cell carcinoma, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer (endometrial cancer), renal cancer (kidney cancer), renal Cell Carcinoma (RCC), prostate cancer, hormone refractory prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer (cervical cancer), gastric cancer (cancer), bladder cancer, hepatoma, breast cancer, colon cancer, head and neck cancer, gastric cancer (gastric cancer), germ cell tumors, pediatric sarcoma, sinus natural killer cells melanoma, bone cancer, skin cancer, uterine cancer, anal region cancer, testicular cancer, fallopian tube cancer, endometrial cancer (carcinoma of the endometrium), cervical cancer (carcinoma of the cervix), vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, rectal cancer, childhood solid tumor, ureteral cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma (squamous cell cancer), environmentally-induced cancers including asbestos-induced cancers, virus-related cancers, or cancers of viral origin (e.g., human papillomavirus (HPV-associated or derived tumor)), and any combination thereof. In certain aspects, the subject has received one, two, three, four, five or more prior cancer treatments. In other aspects, the subject is untreated. In some aspects, the subject develops in the course of other cancer treatments. In certain aspects, the prior cancer treatment comprises immunotherapy. In other aspects, the prior cancer treatment comprises chemotherapy. In some aspects, the tumor has relapsed. In some aspects, the tumor is metastatic. In other aspects, the tumor is not metastatic. In some aspects, the tumor is locally advanced.

In some aspects, the subject has received prior therapy to treat the tumor and the tumor is recurrent or refractory. In certain aspects, the at least one prior therapy comprises a standard of care therapy. In some aspects, the at least one prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some aspects, the at least one prior therapy comprises chemotherapy. In some aspects, the subject has received prior immune-oncology (I-O) therapy to treat the tumor and the tumor is recurrent or refractory. In some aspects, the subject has received more than one prior therapy to treat the tumor and the subject is relapsed or refractory. In other aspects, the subject has received anti-PD-1 or anti-PD-L1 antibody therapy.

In some aspects, the prior therapy line comprises chemotherapy. In some aspects, the chemotherapy comprises platinum-based therapy. In some aspects, the platinum-based therapy comprises a platinum-based antineoplastic agent selected from the group consisting of: cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate (phenanthriplatin), picoplatin, satraplatin, and any combination thereof. In certain aspects, the platinum-based therapy comprises cisplatin. In a particular aspect, the platinum-based therapy comprises carboplatin.

In some aspects, the at least one prior therapy is selected from therapies comprising administering an anticancer agent selected from the group consisting of: platinum agents (e.g., cisplatin, carboplatin), taxane agents (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab)ErlotinibCrizotinib->Cetuximab->And any combination thereof. In certain aspects, the at least one prior therapy comprises platinum-based dual drug chemotherapy.

In some aspects, the subject has undergone disease progression following the at least one prior therapy. In certain aspects, the subject has received at least two prior therapies, at least three prior therapies, at least four prior therapies, or at least five prior therapies. In certain aspects, the subject has received at least two prior therapies. In one aspect, the subject has undergone disease progression following the at least two prior therapies. In certain aspects, the at least two previous therapies comprise a first previous therapy and a second previous therapy, wherein the subject has undergone disease progression after the first previous therapy and/or the second previous therapy, and wherein the first previous therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof; and wherein the second prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy, or any combination thereof. In some aspects, the first prior therapy comprises platinum-based dual drug chemotherapy and the second prior therapy comprises single agent chemotherapy. In certain aspects, the single agent chemotherapy comprises docetaxel.

In certain aspects, the tumor is a PD-L1 positive tumor. As used herein, "PD-L1 positive" may be used interchangeably with "at least about 1% PD-L1 expression". PD-L1 expression may be measured by any method known in the art. In some aspects, PD-L1 expression is measured by automated IHC. A PD-L1 positive tumor may thus have at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of PD-L1 expressing tumor cells as measured by automated IHC. In certain aspects, "PD-L1 positive" means that there are at least 100 cells expressing PD-L1 on the cell surface.

To assess PD-L1 expression, in one aspect, a test tissue sample is obtained from a patient in need of the therapies disclosed herein. In another aspect, the assessment of PD-L1 expression is accomplished without obtaining a test tissue sample. In some aspects, selecting a suitable patient comprises (i) optionally providing a test tissue sample obtained from a patient having a tissue cancer, the test tissue sample comprising tumor cells and/or tumor-infiltrating inflammatory cells; and (ii) assessing the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample based on an assessment that the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample is above a predetermined threshold level.

However, in any method including measuring PD-L1 expression in a test tissue sample, it is understood that the step of providing a test tissue sample obtained from a patient is an optional step. It will also be appreciated that in certain aspects, the "measuring" or "assessing" step for identifying cells expressing PD-L1 (e.g., expression of PD-L1 on the cell surface) or determining the number or proportion of such cells in a test tissue sample is performed by a transformation method that determines PD-L1 expression, such as by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In certain other aspects, no transformation step is involved and PD-L1 expression is assessed by, for example, reviewing reports of test results from a laboratory. In certain aspects, until (and including) the steps of the method of assessing PD-L1 expression provide an intermediate result, the intermediate result may be provided to a physician or other healthcare provider for use in selecting candidates suitable for anti-PD-1 antibodies or anti-PD-L1 antibody therapies. In certain aspects, the step of providing the intermediate result is performed by a medical practitioner or a person acting under the direction of the medical practitioner. In other aspects, these steps are performed by a separate laboratory or by a separate person (e.g., a laboratory technician).

In certain aspects of any of the methods of the invention, the proportion of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In other aspects, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization, or RNase protection. In other aspects, the proportion of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide. In other aspects, the presence of the PD-L1 polypeptide is determined by Immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo imaging or flow cytometry. In some aspects, PD-L1 expression is determined by IHC. In other aspects of all of these methods, cell surface expression of PD-L1 is determined using, for example, IHC or in vivo imaging.

Imaging techniques provide an important tool in cancer research and treatment. Recent developments in molecular imaging systems, including Positron Emission Tomography (PET), single Photon Emission Computed Tomography (SPECT), fluorescence Reflectance Imaging (FRI), fluorescence Mediated Tomography (FMT), bioluminescence imaging (BLI), laser Scanning Confocal Microscopy (LSCM), and multiphoton microscopy (MPM), may presuppose more use of these techniques in cancer research. Some of these molecular imaging systems allow not only the clinician to see the location of the tumor in the body, but also to visualize the expression and activity of specific molecules, cells and biological processes that affect the behavior and/or reactivity of the tumor against therapeutic drugs (condeleis and weissler, "In vivo imaging in cancer," Cold Spring harb.perselect.biol.2 (12): a003848 (2010)). Antibody specificity is coupled with the sensitivity and resolution of PET, making immunoPET imaging particularly attractive for monitoring and determining antigen expression in tissue samples (McCabe and Wu, "Positive progress in immunoPET-not just a coincidence," Cancer biother. Radiopharm.25 (3): 253-61 (2010); olafsen et al, "ImmunoPET imaging of B-cell lymphoma using 124I-anti-CD20 scFv diodes (diabodies)," Protein Eng. Des. Sel.23 (4): 243-9 (2010)). In certain aspects of any of the methods of the invention, PD-L1 expression is determined by immunopet imaging. In certain aspects of any of the methods of the invention, the proportion of cells expressing PD-L1 in the test tissue sample is assessed by performing an assay to determine the presence of PD-L1 polypeptide on the cell surface in the test tissue sample. In certain aspects, the test tissue sample is an FFPE tissue sample. In other aspects, the presence of the PD-L1 polypeptide is determined by IHC assay. In a further aspect, the IHC assay is performed using an automated process. In some aspects, IHC assays are performed using anti-PD-L1 monoclonal antibodies that bind to PD-L1 polypeptides.

In one aspect of the methods of the invention, an automated IHC method is used to determine PD-L1 expression on the surface of cells in FFPE tissue samples. The present disclosure provides methods for detecting the presence of human PD-L1 antigen in a test tissue sample, or quantifying the level of human PD-L1 antigen or the proportion of cells expressing the antigen in a sample, the methods comprising contacting the test sample and a negative control sample with a monoclonal antibody that specifically binds human PD-L1 under conditions that allow for the formation of a complex between the antibody or portion thereof and human PD-L1. In certain aspects, the test and control tissue samples are FFPE samples. Complex formation is then detected, wherein a difference in complex formation between the test sample and the negative control sample is indicative of the presence of human PD-L1 antigen in the sample. PD-L1 expression was quantified using a variety of methods.

In a particular aspect, the automated IHC method comprises: (a) Dewaxing and rehydrating the mounted tissue sections in an automatic staining machine; (b) Antigen was recovered using a visualization chamber (decloaking chamber) and pH 6 buffer (heated to 110 ℃ for 10 min); (c) placing a reagent on the automated staining machine; and (d) a step of operating the autostainer to include neutralizing endogenous peroxidases in the tissue sample; blocking non-specific protein binding sites on the slide; incubating the slide with a primary antibody; incubation with a primary post-staining (postprimary) blocking agent; incubation with NovoLink polymer; adding a chromogen substrate and developing; and counterstained with hematoxylin.

For evaluation of PD-L1 expression in tumor tissue samples, pathologists under a microscopeExamining the film PD-L1 in each field of view ⁺⁺ The number of tumor cells and the percentage of cells positive in the mind were estimated and then averaged to give the final percentage. Different staining intensities were defined as 0/negative, l+/weak, 2+/neutral 3+/strong. Typically, the percentage values are assigned to 0 and 3+ buckets (bins) first, then the intermediate 1+ and 2+ intensities are considered. For highly heterogeneous tissues, the samples are divided into multiple regions, and each region is scored separately and then combined into a single set of percentage values. The percentage of negative and positive cells of different staining intensity was determined from each zone and the median value for each zone was given. The final percentage value of the tissue is given for each of the following staining intensity categories: negative, 1+, 2+ and 3+. It is desirable that the sum of all staining intensities be 100%. In one aspect, the threshold number of cells that are required to be positive for PD-L1 is at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200 cells. In certain aspects, it is desirable that the threshold number of cells that are positive for PD-L1 be at least about 100 cells.

Staining was also assessed in tumor-infiltrating inflammatory cells (e.g., macrophages and lymphocytes). In most cases, macrophages act as an internal positive control, as staining is observed in most macrophages. Although staining of 3+ intensity is not required, macrophages should not be taken into account to rule out any technical failure. Plasma membrane staining of macrophages and lymphocytes was assessed and only positive or negative for all samples were recorded for each cell class. Staining was also characterized by external/internal tumor immune cell name. By "internal" is meant that the immune cells are within the tumor tissue and/or at the boundary of the tumor area without physical insertion between the tumor cells. By "external" is meant that there is no physical association with the tumor and immune cells are found at the periphery associated with connective tissue or any related adjacent tissue.

In some aspects of these scoring methods, samples are scored by two independently working pathologists, followed by combining the scores. In certain other aspects, the identification of positive and negative cells is scored using appropriate software.

Tissue scores (also described as H scores) are used as a more quantitative measure of IHC data. The tissue score was calculated as follows:

Tissue score = [ (tumor% x 1 (low intensity)) + (tumor% x 2 (medium intensity)) + (tumor% x 3 (high intensity) ]

To determine tissue scores, pathologists estimate the percentage of stained cells in each intensity class within the sample. Because the expression of most biomarkers is heterogeneous, the tissue score is a more realistic representation of the overall expression. The final tissue score ranged from 0 (no expression) to 300 (maximum expression).

An alternative means of quantifying PD-L1 expression in a test tissue sample IHC is to determine an Adjusted Inflammation Score (AIS), which is defined as the inflammatory density multiplied by the percentage of PD-L1 expression of tumor-infiltrating inflammatory cells (Taube et al, "Colocalization of inflammatory response with B-h 1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape," sci. Transl. Med.4 (127): 127ra37 (2012).

Compositions of the present disclosure

Pharmaceutical compositions comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are also within the scope of the present disclosure. In some aspects, the pharmaceutical composition is formulated for subcutaneous administration according to the methods disclosed herein. In some aspects, the pharmaceutical compositions are configured such that they exhibit improved properties compared to compositions without both antioxidants or at least one antioxidant. The therapeutic agents of the present disclosure may constitute compositions, e.g., pharmaceutical compositions, containing an antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion), while the carrier for the antibody and/or cytokine-containing composition is suitable for parenteral (e.g., oral) administration. In certain aspects, the pharmaceutical composition does not comprise hyaluronidase.

In some aspects, the pharmaceutical composition comprises at least about 10mg/mL to at least about 500mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects of the present invention, the pharmaceutical composition comprises at least about 10mg/mL to at least about 500mg/mL, at least about 10mg/mL to at least about 400mg/mL, at least about 20mg/mL to at least about 500mg/mL, at least about 20mg/mL to at least about 300mg/mL, at least about 20mg/mL to at least about 200mg/mL, at least about 10mg/mL to at least about 190mg/mL, at least about 180mg/mL, at least about 10mg/mL to at least about 170mg/mL, at least about 10mg/mL to at least about 160mg/mL, at least about 10mg/mL to at least about 150mg/mL, at least about 20mg/mL to at least about 500mg/mL, at least about 20mg/mL to at least about 250mg/mL, at least about 20mg/mL to at least about 200mg/mL, at least about 20mg/mL to at least about 190mg/mL, at least about 180mg/mL to at least about 180mg/mL, at least about 180mg/mL to at least about 200mg/mL, at least about 150mg/mL to at least about 150mg/mL, at least about 20mg/mL to at least about 20mg/mL, at least about 20mg/mL to at least about 250mg/mL, at least about 250 mg/mg to at least, at least about 100mg/mL to at least about 130mg/mL or at least about 108mg/mL to at least about 132mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 50mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 60mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 70mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 75mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 80mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 90mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 100mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 108mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 110mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 120mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 130mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 132mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 135mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 140mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 150mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 160mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 170mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 175mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 180mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 190mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody. In some aspects, the pharmaceutical composition comprises at least about 200mg/mL of the anti-PD-1 antibody or the anti-PD-L1 antibody.

III.A. endoglycosidase hydrolase

In some aspects, the pharmaceutical composition comprises an endoglycosidase hydrolase. In some aspects, the pharmaceutical composition comprises at least about 50U/mL to at least about 48000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 50U/mL to at least about 5000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 50U/mL, at least about 100U/mL, at least about 150U/mL, at least about 200U/mL, at least about 250U/mL, at least about 300U/mL, at least about 350U/mL, at least about 400U/mL, at least about 450U/mL, at least about 500U/mL, at least about 750U/mL, at least about 1000U/mL, at least about 1500U/mL, at least about 2000U/mL, at least about 2500U/mL, at least about 3000U/mL, at least about 3500U/mL, at least about 4000U/mL, at least about 4500U/mL, at least about 5000U/mL, at least about 5500U/mL, at least about 6000U/mL, at least about 6500U/mL, at least about 7000U/mL, at least about 7500U/mL, at least about 8000U/mL, at least about 8500U/mL, at least about 9000U/mL, at least about 9500U/mL, at least about 10,000U/mL, endoglycosidase hydrolase (e.g., hurPH 20). In some aspects, the pharmaceutical composition comprises at least about 500U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 1000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 2000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 2500U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 3000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 3500U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 4000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 4500U/mL endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 5000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 6000U/mL of endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 7000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 8000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 9000U/mL of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 10,000u/mL of an endoglycosidase hydrolase (e.g., rHuPH 20).

In some aspects, the pharmaceutical composition comprises at least about 50 units to at least about 100,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 50 units, at least about 100 units, at least about 150 units, at least about 200 units, at least about 250 units, at least about 300 units, at least about 400 units, at least about 500 units, at least about 600 units, at least about 700 units, at least about 800 units, at least about 900 units, at least about 1000 units, at least about 1500 units, at least about 2000 units, at least about 2500 units, at least about 3000 units, at least about 4000 units, at least about 5000 units, at least about 10,000 units, at least about 15,000 units, at least about 20,000 units, at least about 25,000 units, at least about 30,000 units, at least about 35,000 units, at least about 40,000 units, at least about 45,000 units, at least about 48,000 units, at least about 50,000 units, at least about 55,000 units, at least about 60,000 units, at least about 65,000 units, at least about 70,000 units, at least about 75,000 units, at least about 80,000 units, at least about 85,000 units, at least about 90,000 units, at least about 100,000 units, or at least about 95,000 units of an endoglycosidase (e.g., endoglycosidase). In some aspects, the pharmaceutical composition comprises at least about 20,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 30,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 40,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 50,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 60,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 70,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 80,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 90,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20). In some aspects, the pharmaceutical composition comprises at least about 100,000 units of an endoglycosidase hydrolase (e.g., rHuPH 20).

Those skilled in the art will readily appreciate that the amount of the endoglycosidase hydrolase (e.g., rHuPH 20) may be expressed in units or U/mL, or the amount of the endoglycosidase hydrolase (e.g., rHuPH 20) may be expressed in mg/mL (or in other weight-based units). For example, in some aspects, the pharmaceutical composition comprises an endoglycosidase hydrolase (e.g., rHuPH 20) in an amount expressed as at least about 500U/mL or at least about 0.00455 mg/mL. In another example, in some aspects, the pharmaceutical composition comprises an endoglycosidase hydrolase (e.g., rHuPH 20) in an amount expressed as at least about 2000U/mL or at least about 0.0182 mg/mL.

In certain aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20.

In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20.

In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 2000U/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) About 5mM methionine, and (g) about 0.0182mg/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises (a) about 672mg of nivolumab; (b) about 8.68mg L-histidine; (c) about 11.8mg histidine HCl H2O; (d) about 479mg sucrose; (e) about 2.80mg polysorbate 80; (f) about 0.110mg of pentetic acid; (g) about 4.18mg methionine; (h) about 0.102mg rhuph20; wherein (a) - (h) are reconstituted in water to a final volume of at least about 5.6 mL.

III.B. antioxidants

In some aspects, the pharmaceutical composition further comprises an antioxidant. Any antioxidant may be used in the pharmaceutical compositions disclosed herein. In some aspects, the antioxidant is selected from methionine, tryptophan, and histidine, cysteine, ascorbic acid, glycine, pentetic acid (DTPA), and EDTA. In certain aspects, the pharmaceutical composition comprises methionine. In some aspects, the pharmaceutical composition comprises at least two antioxidants. In some aspects, more than one antioxidant (e.g., two antioxidants) prevents oxidation of formulation components and/or improves the stability of the antibody. In some aspects, the at least two antioxidants comprise (i) methionine and EDTA, or (ii) methionine and pentetic acid (DTPA).

In some aspects, the pharmaceutical composition comprises the anti-PD-1 antibody, methionine, and pentetic acid (DTPA). In some aspects, the pharmaceutical composition comprises at least about 0.1mM to at least about 100mM methionine. In some aspects, the pharmaceutical composition comprises at least about 1mM, at least about 1.5mM, at least about 2mM, at least about 2.5mM, at least about 3mM, at least about 3.5mM, at least about 4mM, at least about 4.5mM, at least about 5mM, at least about 5.5mM, at least about 6mM, at least about 6.5mM, at least about 7mM, at least about 7.5mM, at least about 8mM, at least about 8.5mM, at least about 9mM, at least about 9.5mM, or at least about 10mM, at least about 11mM, at least about 12mM, at least about 13mM, at least about 14mM, at least about 15mM, at least about 16mM, at least about 17mM, at least about 18mM, at least about 19mM, or at least about 20mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 10mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 9mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 8mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 7mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 6mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 5mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 4mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 3mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 2mM methionine. In certain aspects, the pharmaceutical composition comprises at least about 1mM methionine.

In some aspects, the pharmaceutical composition comprises at least about 1mM to at least about 250mM pentetic acid (DTPA). In some aspects, the pharmaceutical composition comprises at least about 1 μΜ, at least about 5 μΜ, at least about 10 μΜ, at least about 15 μΜ, at least about 20 μΜ, at least about 25 μΜ, at least about 30 μΜ, at least about 35 μΜ, at least about 40 μΜ, at least about 45 μΜ, at least about 50 μΜ, at least about 55 μΜ, at least about 60 μΜ, at least about 65 μΜ, at least about 70 μΜ, at least about 75 μΜ, at least about 80 μΜ, at least about 85 μΜ, at least about 90 μΜ, at least about 95 μΜ or at least about 100 μΜ, at least about 110 μΜ, at least about 120 μΜ, at least about 130 μΜ, at least about 140 μΜ, at least about 150 μΜ, at least about 160 μΜ, at least about 170 μΜ, at least about 180 μΜ, at least about 190 μΜ or at least about 200 μΜ DTPA. In certain aspects, the pharmaceutical composition comprises at least about 75 μm pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 70 μΜ pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 65 μm pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 60 μΜ pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 55 μΜ pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 50 μm pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 45 μm pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 40 μm pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 35 μΜ pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 30 μm pentetic acid (DTPA). In certain aspects, the pharmaceutical composition comprises at least about 25 μm pentetic acid (DTPA).

III.C. tonicity regulator/stabilizer

In some aspects, the pharmaceutical composition further comprises a tonicity modifier and/or stabilizer. Any tonicity modifier and/or any stabilizer may be used in the pharmaceutical compositions disclosed herein. In some aspects, the tonicity modifier and/or stabilizer includes a sugar, an amino acid, a polyol, a salt, or any combination thereof. In some aspects, the tonicity modifier and/or stabilizer is selected from sucrose, sorbitol, trehalose, mannitol, glycerol, glycine, leucine, isoleucine, sodium chloride, proline, arginine, polyols, amino acids and salts.

In certain aspects, the pharmaceutical composition comprises sucrose. In some aspects, the pharmaceutical composition comprises at least about 1mM to at least about 500mM sucrose. In some aspects, the pharmaceutical composition comprises at least about 10mM to at least about 400mM, at least about 50mM to at least about 400mM, at least about 100mM to at least about 400mM, at least about 150mM to at least about 400mM, at least about 200mM to at least about 400mM, at least about 250mM to at least about 400mM, at least about 300mM to at least about 400mM, at least about 350mM to at least about 400mM, at least about 50mM to at least about 350mM, at least about 100mM to at least about 300mM, at least about 100mM to at least about 250mM, at least about 100mM to at least about 200mM, at least about 100mM to at least about 150mM, at least about 200mM to at least about 400mM, at least about 200mM to at least about 300mM sucrose, or at least about 200mM to at least about 250mM. In some aspects, the pharmaceutical composition comprises at least about 10mM, at least about 20mM, at least about 30mM, at least about 40mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, at least about 100mM, at least about 110mM, at least about 120mM, at least about 130mM, at least about 140mM, at least about 150mM, at least about 160mM, at least about 170mM, at least about 180mM, at least about 190mM, at least about 200mM, at least about 210mM, at least about 220mM, at least about 230mM, at least about 240mM, at least about 250mM, at least about 260mM, at least about 270mM, at least about 280mM, at least about 290mM, at least about 300mM, at least about 310mM, at least about 320mM, at least about 330mM, at least about 340mM, at least about 350mM, at least about 360mM, at least about 370mM, at least about 380mM, at least about 400mM, at least about 410mM, at least about 420mM, at least about 430mM, at least about 450mM, at least about 460mM, at least about 470mM, at least about 500mM, or at least about 500 mM. In certain aspects, the pharmaceutical composition comprises at least about 200mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 210mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 220mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 230mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 240mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 250mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 260mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 270mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 280mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 290mM sucrose. In certain aspects, the pharmaceutical composition comprises at least about 300mM sucrose.

III.D. buffering agents

In some aspects, the pharmaceutical composition further comprises a buffer. In some aspects, the buffer is selected from histidine, succinate, tromethamine, sodium phosphate, sodium acetate, and sodium citrate. In certain aspects, the pharmaceutical composition comprises histidine. In certain aspects, the pharmaceutical composition comprises citrate. In some aspects, the pharmaceutical composition comprises at least about 1mM to at least about 100mM histidine. In some aspects, the pharmaceutical composition comprises at least about 5mM to at least about 100mM, at least about 10mM to at least about 100mM, at least about 15mM to at least about 100mM, at least about 20mM to at least about 100mM, at least about 25mM to at least about 100mM, at least about 30mM to at least about 100mM, at least about 35mM to at least about 100mM, at least about 40mM to at least about 100mM, at least about 45mM to at least about 100mM, at least about 50mM to at least about 100mM, at least about 10mM to at least about 75mM, at least about 10mM to at least about 50mM, at least about 10mM to at least about 40mM, at least about 10mM to at least about 30mM, at least about 15mM to at least about 30mM, at least about 10mM to at least about 25mM, or at least about 15mM to at least about 25mM histidine.

In some aspects, the pharmaceutical composition comprises at least about 5mM, at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, or at least about 100mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 10mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 15mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 20mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 25mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 30mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 35mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 40mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 45mM histidine. In certain aspects, the pharmaceutical composition comprises at least about 50mM histidine.

In some aspects, the pharmaceutical composition comprises a pH of about 5.2 to about 6.8. In some aspects, the pharmaceutical composition has a pH of about 5.2. In some aspects, the pharmaceutical composition has a pH of about 5.3. In some aspects, the pharmaceutical composition has a pH of about 5.4. In some aspects, the pharmaceutical composition has a pH of about 5.5. In some aspects, the pharmaceutical composition has a pH of about 5.6. In some aspects, the pharmaceutical composition has a pH of about 5.7. In some aspects, the pharmaceutical composition has a pH of about 5.8. In some aspects, the pharmaceutical composition has a pH of about 5.9. In some aspects, the pharmaceutical composition has a pH of about 6.0. In some aspects, the pharmaceutical composition has a pH of about 6.1. In some aspects, the pharmaceutical composition has a pH of about 6.2. In some aspects, the pharmaceutical composition has a pH of about 6.3. In some aspects, the pharmaceutical composition has a pH of about 6.4. In some aspects, the pharmaceutical composition has a pH of about 6.5. In some aspects, the pharmaceutical composition has a pH of about 6.6. In some aspects, the pharmaceutical composition has a pH of about 6.7. In some aspects, the pharmaceutical composition has a pH of about 6.8.

III.E. surfactants

In some aspects, the pharmaceutical composition further comprises a surfactant. Any surfactant may be used in the pharmaceutical compositions disclosed herein. In some aspects, the surfactant is selected from polysorbate 20, polysorbate 80, and poloxamer 188. In certain aspects, the pharmaceutical composition comprises polysorbate 80. In some aspects, the pharmaceutical composition comprises at least about 0.001% to at least about 1% w/v polysorbate 80. In some aspects, the pharmaceutical composition comprises at least about 0.01% to at least about 0.1%, at least about 0.02% to at least about 0.1%, at least about 0.03% to at least about 0.1%, at least about 0.04% to at least about 0.1%, at least about 0.05% to at least about 0.1%, at least about 0.01% to at least about 0.09%, at least about 0.01% to at least about 0.8%, at least about 0.01% to at least about 0.7%, at least about 0.01% to at least about 0.6%, at least about 0.01% to at least about 0.5%, at least about 0.02% to at least about 0.09%, at least about 0.03% to at least about 0.08%, at least about 0.04% to at least about 0.07%, or at least about 0.04% to at least about 0.06% w/v polysorbate 80. In some aspects, the pharmaceutical composition comprises at least about 0.01% to at least about 0.1% w/v polysorbate 80.

In some aspects, the pharmaceutical composition comprises at least about 0.01% w/v, at least about 0.02% w/v, at least about 0.03% w/v, at least about 0.04% w/v, at least about 0.05% w/v, at least about 0.06% w/v, at least about 0.07% w/v, at least about 0.08% w/v, at least about 0.09% w/v, or at least about 0.1% w/v polysorbate 80. In certain aspects, the pharmaceutical composition comprises at least about 0.03% w/v polysorbate 80. In certain aspects, the pharmaceutical composition comprises at least about 0.04% w/v polysorbate 80. In certain aspects, the pharmaceutical composition comprises at least about 0.05% w/v polysorbate 80. In certain aspects, the pharmaceutical composition comprises at least about 0.06% w/v polysorbate 80. In certain aspects, the pharmaceutical composition comprises at least about 0.07% w/v polysorbate 80.

In certain aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine.

In some aspects, the pharmaceutical composition comprises: (a) an anti-PD-1 antibody, e.g., nivolumab; (b) checkpoint inhibitors, e.g., anti-CTLA-4 antibodies; (c) about 20mM histidine; (d) about 250mM sucrose; (e) about 0.05% w/v polysorbate 80; (f) about 50 μm pentetic acid; and (g) about 5mM methionine. In some aspects, the pharmaceutical composition comprises: (a) an anti-PD-1 antibody, e.g., nivolumab; (b) checkpoint inhibitors, e.g., anti-CTLA-4 antibodies; (c) about 20mM histidine; (d) about 250mM sucrose; (e) about 0.05% w/v polysorbate 80; (f) about 50 μm pentetic acid; (g) about 5mM methionine; and (h) about 2000U/mL rHuPH20.

In some aspects, the pharmaceutical composition comprises: (a) an anti-PD-1 antibody, e.g., nivolumab; (b) a checkpoint inhibitor, e.g., an anti-LAG-3 antibody; (c) about 20mM histidine; (d) about 250mM sucrose; (e) about 0.05% w/v polysorbate 80; (f) about 50 μm pentetic acid; and (g) about 5mM methionine. In some aspects, the pharmaceutical composition comprises: (a) an anti-PD-1 antibody, e.g., nivolumab; (b) a checkpoint inhibitor, e.g., an anti-LAG-3 antibody; (c) about 20mM histidine; (d) about 250mM sucrose; (e) about 0.05% w/v polysorbate 80; (f) about 50 μm pentetic acid; (g) about 5mM methionine; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the unit doses described herein comprise: (a) about 120mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20. In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the unit doses described herein comprise: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises (a) about 672mg of nivolumab; (b) about 8.68mg L-histidine; (c) about 11.8mg histidine HCl H2O; (d) about 479mg sucrose; (e) about 2.80mg polysorbate 80; (f) about 0.110mg of pentetic acid; (g) about 4.18mg methionine; wherein (a) - (g) are reconstituted in water (e.g., sterile water for injection, SWFI) to a final volume of at least about 5.6 mL.

III.F. containers and delivery devices

Some aspects of the present disclosure relate to a vial comprising a pharmaceutical composition disclosed herein. In some aspects, the vial comprises a unit dose of the pharmaceutical composition. In some aspects, the vial comprises (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In some aspects, the vial does not comprise hyaluronidase.

In some aspects, the vial is a syringe. Any syringe may be used in the compositions and methods disclosed herein. In some aspects, the syringe contains one or more mechanical elements that improve subcutaneous administration.

In some aspects, the vial is an auto-injector. Typically, automatic injectors are operated by the patient simply by applying pressure on the injection site to actuate the needle and subsequent drug flow. The pressure causes the needle shield to actuate which engages the needle and causes the device to inject the medicament. Accordingly, some aspects of the present disclosure relate to an auto-injector comprising (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In some aspects, the auto-injector does not comprise hyaluronidase.

In some aspects, the vial is a pen injector. Standard pen injectors require a patient activation button that pushes the needle into the targeted injection site. Accordingly, some aspects of the present disclosure relate to an injection pen comprising (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In some aspects, the injection pen does not comprise hyaluronidase.

In some aspects, the vial is a wearable pump or wearable device. In some aspects, the wearable pump is a patch pump (patch pump). Accordingly, some aspects of the present disclosure relate to a wearable pump comprising (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In some aspects, the wearable pump does not comprise hyaluronidase.

Some aspects of the disclosure relate to an auto-injector comprising (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 0.0182mg/mL rHuPH20.

In some aspects, the vial is a pen injector. Standard pen injectors require a patient activation button that pushes the needle into the targeted injection site. Accordingly, some aspects of the present disclosure relate to an injection pen comprising (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 0.0182mg/mL rHuPH20.

In some aspects, the vial is a wearable pump or wearable device. In some aspects, the wearable pump is a patch pump (patch pump). Accordingly, some aspects of the present disclosure relate to a wearable pump comprising (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM L-histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 0.0182mg/mL rHuPH20.

III.G. additional therapeutic agents

In some aspects, the pharmaceutical composition further comprises a second therapeutic agent (e.g., an anti-PD-1 antibody and an additional therapeutic agent, or an anti-PD-L1 antibody and an additional therapeutic agent). In some aspects, the pharmaceutical composition further comprises a third therapeutic agent. The additional therapeutic agent may include any therapy for treating a tumor in a subject and/or any standard of care therapy, as disclosed herein. In some aspects, the additional therapeutic agent comprises a second antibody. In some aspects, the additional therapeutic agent comprises an antibody or antigen-binding portion thereof that specifically binds CTLA-4, LAG-3, TIGIT, TIM3, NKG2a, OX40, ICOS, MICA, CD137, KIR, tgfβ, IL-10, IL-8, B7-H4, fas ligand, CXCR4, mesothelin, CD27, GITR, or any combination thereof. In some aspects, the second therapeutic agent, the third therapeutic agent, or both comprise IL-2 (e.g., bei Peia interleukin). In some aspects, the second therapeutic agent, the third therapeutic agent, or both comprise IL12-Fc (e.g., BMS-986415).

In some aspects, the antibody is a multispecific antibody. In some aspects, the antibody is a bispecific antibody. In some aspects, the antibody is a trispecific antibody. In some aspects, the antibody specifically binds (i) PD-1 and (ii) a second antigen. In some aspects, the antibody specifically binds (i) PD-1, (ii) a second antigen, and (iii) a third antigen. In some aspects, the antibody specifically binds (i) PD-L1 and (ii) a second antigen. In some aspects, the antibody specifically binds (i) PD-L1, (ii) a second antigen, and (iii) a third antigen. In some aspects, the second antigen and the third antigen are the same. In some aspects, the second antigen and the third antigen are different. In some aspects, the second antigen is CD3.

In some aspects, the antibody specifically binds to (i) TIGIT and (ii) an inhibitory receptor expressed on T cells, NK cells, or both. In some aspects, the antibody specifically binds to (i) CD40 and (ii) CD20.

In some aspects, the second antibody comprises an anti-CTLA-4 antibody. The anti-CTLA-4 antibody can be any antibody or antigen-binding portion thereof that binds CTLA-4 and inhibits its activity. In some aspects, the anti-CTLA-4 antibody is any anti-CTLA-4 antibody disclosed herein. In some aspects, the second antibody comprises tremelimumab. In some aspects, the second antibody comprises ipilimumab.

In some aspects, the second antibody comprises an anti-LAG 3 antibody. The anti-LAG 3 antibody may be any antibody or antigen-binding portion thereof that binds LAG-3 and inhibits its activity. In some aspects, the anti-LAG 3 antibody comprises any anti-LAG 3 antibody disclosed herein. In some aspects, the second antibody comprises 25F7.

In some aspects, the second antibody comprises an anti-CD 137 antibody. The anti-CD 137 antibody may be any antibody or antigen-binding portion thereof that binds CD137 and inhibits its activity. In some aspects, the anti-CD 137 antibodies include any of the anti-CD 137 antibodies disclosed herein. In some aspects, the second antibody comprises nivolumab.

In some aspects, the second antibody comprises an anti-KIR antibody. The anti-KIR antibodies include any antibody or antigen-binding portion thereof that binds KIR and inhibits its activity. In some aspects, the anti-KIR antibodies comprise any anti-KIR antibody disclosed herein. In some aspects, the second antibody comprises Li Ruilu mab.

In some aspects, the second antibody comprises an anti-GITR antibody. The anti-GITR antibody may be any antibody or antigen-binding portion thereof that binds GITR and inhibits its activity. In some aspects, the anti-GITR antibody comprises any anti-GITR antibody disclosed herein. In some aspects, the second antibody comprises MK4166. In some aspects, the second antibody comprises TRX518.

In some aspects, the second antibody comprises an anti-CD 96 antibody. In some aspects, the second antibody comprises an anti-TIM 3 antibody. In some aspects, the second antibody comprises an anti-VISTA antibody. In some aspects, the second antibody comprises an anti-NKG 2a antibody. In some aspects, the second antibody comprises an anti-ICOS antibody. In some aspects, the second antibody comprises an anti-OX 40 antibody. In some aspects, the second antibody comprises an anti-IL 8 antibody, e.g-IL8(BMS-986253)。

III.G.1. Anti-CTLA-4 antibodies

In some aspects, the second antibody comprises an anti-CTLA-4 antibody. anti-CTLA-4 antibodies known in the art can be used in the compositions and methods of the present disclosure. The anti-CTLA-4 antibodies of the invention bind to human CTLA-4, thereby disrupting the interaction of CTLA-4 with human B7 receptor. Since the interaction of CTLA-4 with B7 transduces a signal that leads to inactivation of T cells carrying CTLA-4 receptor, disruption of the interaction effectively induces, enhances or extends activation of such T cells, thereby inducing, enhancing or extending an immune response.

Human monoclonal antibodies that specifically bind to CTLA-4 with high affinity have been disclosed in U.S. patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies have been described, for example, in the following: U.S. Pat. nos. 5,977,318, 6,051,227, 6,682,736 and 7,034,121, international publication nos. WO 2012/12244, WO 2007/113648, W O2016/196237 and WO 2000/037504, each of which is incorporated herein by reference in its entirety. The anti-CTLA-4 human monoclonal antibody disclosed in us patent No. 6,984,720 has been demonstrated to exhibit one or more of the following characteristics: (a) Specific binding to human CTLA-4, the binding affinity of which is reflected by equilibrium association constant (K _a ) At least about 10 ⁷ M ^-1 Or about 10 ⁹ M ^-1 Or about 10 ¹⁰ M ^-1 To 10 ¹¹ M ^-1 Or higher, as determined by Biacore analysis; (b) Kinetic association constant (k) _a ) At least about 10 ³ About 10 ⁴ Or about 10 ⁵ m ^-1 s ^-1 The method comprises the steps of carrying out a first treatment on the surface of the (c) Kinetic dissociation constant (k) _d ) At least about 10 ³ About 10 ⁴ Or about 10 ⁵ m ^-1 s ^-1 The method comprises the steps of carrying out a first treatment on the surface of the And (d) inhibiting binding of CTLA-4 to B7-1 (CD 80) and B7-2 (CD 86). anti-CTLA-4 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the foregoing characteristics.

In certain aspects, the CTLA-4 antibody is selected from ipilimumab (also known asMDX-010, 10D1; see U.S. patent No. 6,984,720), MK1308 (Merck), AGEN-1884 (agalus inc; see WO 2016/196237) and trimethoprim (AstraZeneca; also known as tiximumab, CP-675,206; see WO 2000/037504 and Ribas, update Cancer Ther.2 (3): 133-39 (2007)). In a particular aspect, the anti-CTLA-4 antibody is ipilimumab.

In a particular aspect, the CTLA-4 antibody is ipilimumab for use in the compositions and methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving total survival (OS) in patients with advanced melanoma.

In a particular aspect, the CTLA-4 antibody is tremelimumab. In a particular aspect, the CTLA-4 antibody is MK-1308. In a particular aspect, the CTLA-4 antibody is AGEN-1884.

In some aspects, the CTLA-4 antibody is nonfucosylated or hypofucosylated. In some aspects, the CTLA-4 antibodies exhibit enhanced ADCC and/or ADCP activity. In some aspects, the CTLA-4 antibody is BMS-986218, as described in PCT/US 18/19868.

anti-CTLA-4 antibodies useful in the disclosed compositions and methods also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any anti-CTLA-4 antibody disclosed herein (e.g., ipilimumab and/or tremelimumab) for binding to human CTLA-4. In some aspects, the anti-CTLA-4 antibody binds to the same epitope as any anti-CTLA-4 antibody described herein (e.g., ipilimumab and/or tremelimumab). The ability of antibodies to cross-compete for binding to an antigen suggests that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have very similar functional properties to the reference antibodies (e.g., ipilimumab and/or tremelimumab) due to their binding to the same epitope region of CTLA-4. Standard CTLA-4 binding assays (such as Biacore assays, ELISA assays or flow cytometry) can be readily identified based on their ability to cross-compete with ipilimumab and/or tremelimumab (see, e.g., WO 2013/173223).

In certain aspects, antibodies that cross-compete with ipilimumab and/or tremelimumab for binding to human CTLA-4 or that bind to the same epitope region of human CTLA-4 antibodies as ipilimumab and/or tremelimumab are monoclonal antibodies. For administration to a human subject, these cross-competing antibodies are chimeric, engineered or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies may be prepared and isolated by methods well known in the art.

anti-CTLA-4 antibodies useful in the compositions and methods of the disclosed invention also include antigen-binding portions of the above antibodies. It is well documented that the antigen binding function of an antibody can be performed by fragments of a full length antibody.

In some aspects, the anti-CTLA-4 antibody can be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CTLA-4 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CTLA-4 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CTLA-4 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CTLA-4 antibody; and (h) about 2000U/mL rHuPH20.

anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and disrupt the interaction of CTLA-4 with human B7 receptors. In any of the compositions or methods disclosed herein, an anti-CTLA-4 "antibody" includes an antigen-binding portion or fragment that binds CTLA-4 and exhibits similar functional properties as an intact antibody in inhibiting CTLA-4 interaction with human B7 receptor and up-regulating the immune system. In certain aspects, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and/or tremelimumab for binding to human CTLA-4.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CTLA-4 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CTLA-4 and (ii) CD3.

III.G.2. Anti-LAG-3 antibodies

In some aspects, the second antibody comprises an anti-LAG-3 antibody, e.g., a rayleigh Li Shan antibody. The anti-LAG-3 antibodies of the present disclosure bind to human LAG-3. Antibodies that bind to LAG-3 have been disclosed in international publication nos. WO/2015/042246 and 2014/0093511 and 2011/0150892, each of which is incorporated herein by reference in its entirety.

An exemplary LAG-3 antibody useful in the present disclosure is 25F7 (described in U.S. publication No. 2011/0150892). Another exemplary LAG-3 antibody useful in the present disclosure is BMS-986016 (Rala Li Shan antibody). In some aspects, anti-LAG-3 antibodies useful in the present disclosure cross-compete with 25F7 or BMS-986016. In some aspects, anti-LAG-3 antibodies useful in the present disclosure bind to the same epitope as 25F7 or BMS-986016. In some aspects, the anti-LAG-3 antibody comprises six CDRs of 25F7 or BMS-986016.

Other art-recognized anti-LAG-3 antibodies that may be used in the methods and/or compositions of the present disclosure include IMP731 (H5L 7 BW) as described in US 2011/007033, WO 2016028672, and MK-4280 (28G-10, favirtuzumab), burova E et al, j.immunother.cancer (2016) as described in US publication 2020/0055938; 4 (supplement 1) P195 and REGN3767 (French mab) described in U.S. Pat. No. 10,358,495, humanized BAP050, GSK2831781, described in WO2017/019894, IMP-701 (LAG 525; era Li Shan antibody), aLAG3 (0414), aLAG3 (0416), sym022, TSR-033, TSR-075, xmAb841 (previously XmAb 22841), MGD013 (Tepolitumab), BI754111, FS118, P13B 02-30, AVA-017, AGEN1746, RO7247669, INCAGN02385, IBI-110, EMB-02, IBI-323, LBL-007 and ABL501. These and other anti-LAG-3 antibodies useful in the claimed invention can be found, for example, in the following documents: US 10,188,730, WO 2016/028672, WO 2017/106129, WO 2017/062888, WO 2009/044273, WO 2018/069500, WO 2016/126858, WO 2014/179664, WO 2016/200782, WO 2015/200119, WO 2017/019846, WO 2017/198741, WO 2017/220555, WO 2017/220569, WO 2018/071500, WO 2017/015560, WO 2017/025498, WO 2017/087589, WO 2017/087901, WO 2018/083087, WO 2017/149743, WO 2017/9995, US 2017/0260271, WO 2017/086367, WO 2017/086419, WO 2018/034227, WO 2018/185046, WO 2018/042170, WO 19/011088, WO 201208180, WO 2012084/208180, WO 2010189 and WO 201018730. The contents of each of these references are incorporated by reference in their entirety.

anti-LAG-3 antibodies that can be used in the methods and/or compositions of the present disclosure also include isolated antibodies that specifically bind to human LAG-3 and cross-compete with any anti-LAG-3 antibody disclosed herein (e.g., a rayleigh Li Shan antibody) for binding to human LAG-3. In some aspects, the anti-LAG-3 antibody binds to the same epitope as any of the anti-LAG-3 antibodies described herein (e.g., a rayleigh Li Shan antibody).

In some aspects, the antibody cross-competes with any anti-LAG-3 antibody disclosed herein (e.g., a rally Li Shan antibody) for binding to human LAG-3 or to the same epitope region as any anti-LAG-3 antibody disclosed herein (e.g., a rally Li Shan antibody) is a monoclonal antibody. For administration to a human subject, these cross-competing antibodies are chimeric, engineered or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies may be prepared and isolated by methods well known in the art.

The ability of an antibody to cross-compete for binding to an antigen suggests that the antibody binds to the same epitope region of the antigen and sterically blocks the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have very similar functional properties to the reference antibody (e.g., the rayleigh Li Shan antibody) because they bind to the same epitope region. They can be readily identified in standard binding assays (such as Biacore assays, ELISA assays or flow cytometry) based on their ability to cross-compete with cross-competing antibodies (see, e.g., WO 2013/173223).

anti-LAG-3 antibodies that can be used in the methods and/or compositions of the present disclosure also include antigen-binding portions of any of the above full-length antibodies. It is well documented that the antigen binding function of an antibody can be performed by fragments of a full length antibody.

In some aspects, the anti-LAG-3 antibody is a full-length antibody.

In some aspects, the anti-LAG-3 antibody is a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, or a multispecific antibody. In some aspects, the multispecific antibody is a amphipathic retargeting antibody (DART), DVD-Ig, or bispecific antibody.

In some aspects, the anti-LAG-3 antibody is a F (ab ') 2 fragment, fab' fragment, fab fragment, fv fragment, scFv fragment, dsFv fragment, dAb fragment, or single chain binding polypeptide.

In some aspects, the anti-LAG-3 antibody is BMS-986016 (Ruila Li Shan antibody), IMP731 (H5L 7 BW), MK4280 (28G-10, favirtuzumab), REGN3767 (Fuolimumab), GSK2831781, humanized BAP050, IMP-701 (LAG 525, ela Li Shan antibody), aLAG3 (0414), aLAG3 (0416), sym022, TSR-033, TSR-075, xmAb841 (XmAb 22841), MGD013 (Tepolimumab), BI754111, FS118, P13B 02-30, AVA-017, 25F7, AGEN1746, RO7247669, INCAGN02385, IBI-110, EMB-02, IBI-323, LBL-007, ABL501, or comprises antigen binding portions thereof.

In some aspects, the anti-LAG-3 antibody is a rale Li Shan antibody.

In some aspects, the anti-LAG-3 antibody is MGD013 (terpolimumab), which is bispecific PD-1 x LAG-3DART.

In some aspects, the anti-LAG-3 antibody is REGN3767 (furilimumab).

In some aspects, the anti-LAG-3 antibody is LAG525 (erla Li Shan antibody).

In some aspects, the anti-LAG-3 antibody is MK4280 (favirzelizumab).

In some aspects, the anti-LAG-3 antibody may be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-LAG-3 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) rale Li Shan resistance. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-LAG-3 antibody; and (h) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) a rilla Li Shan antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-LAG-3 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) rale Li Shan resistance. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-LAG-3 antibody; and (h) about 2000U/mL rHuPH20. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) a rilla Li Shan antibody; and (h) about 2000U/mL rHuPH20.

An exemplary LAG-3 antibody useful in the present disclosure is 25F7 (described in U.S. publication No. 2011/0150892). Another exemplary LAG-3 antibody useful in the present disclosure is BMS-986016. In one aspect, the anti-LAG-3 antibodies useful in the compositions cross-compete with 25F7 or BMS-986016. In another aspect, an anti-LAG-3 antibody useful in the composition binds to the same epitope as 25F7 or BMS-986016. In other aspects, the anti-LAG-3 antibody comprises six CDRs of 25F7 or BMS-986016.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) LAG-3 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) LAG-3 and (ii) CD3.

III.G.3. anti-CD 137 antibodies

In some aspects, the second antibody comprises an anti-CD 137 antibody. anti-CD 137 antibodies specifically bind to and activate CD137 expressing immune cells, thereby stimulating an immune response against tumor cells, particularly a cytotoxic T cell response. Antibodies that bind CD137 have been disclosed in U.S. publication No. 2005/0095244 and U.S. patent nos. 7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685, 6,355,476, 6,362,325, 6,974,863, and 6,210,669.

In some aspects, an anti-CD 137 antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CD 137 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CD 137 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CD 137 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CD 137 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the anti-CD 137 antibody is Ureprunob (BMS-663513), described in U.S. Pat. No. 7,288,638 (20H4.9-IgG 4[10C7 or BMS-663513 ]). In some aspects, the anti-CD 137 antibody is BMS-663031 (20H4.9-IgG 1), described in U.S. Pat. No. 7,288,638. In some aspects, the anti-CD 137 antibody is 4E9 or BMS-554271, described in U.S. patent No. 6,887,673. In some aspects, the anti-CD 137 antibody is U.S. patent No. 7,214,493;6,303,121;6,569,997;6,905,685; or 6,355,476. In some aspects, the anti-CD 137 antibody is 1D8 or BMS-469492;3H3 or BMS-469497; or 3E1, described in U.S. patent No. 6,362,325. In some aspects, the anti-CD 137 antibody is an antibody disclosed in issued U.S. patent No. 6,974,863 (e.g., 53 A2). In some aspects, the anti-CD 137 antibody is an antibody disclosed in issued U.S. patent No. 6,210,669 (e.g., 1D8, 3B8, or 3E 1). In some aspects, the antibody is PF-05082566 (PF-2566) of the Pfizer. In other aspects, the anti-CD 137 antibodies useful in the invention cross-compete with the anti-CD 137 antibodies disclosed herein. In some aspects, the anti-CD 137 antibodies bind to the same epitope as the anti-CD 137 antibodies disclosed herein. In other aspects, an anti-CD 137 antibody useful in the present disclosure comprises the six CDRs of an anti-CD 137 antibody disclosed herein.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CD137 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CD137 and (ii) CD3.

III.G.4. anti-KIR antibodies

In some aspects, the second antibody comprises an anti-KIR 3 antibody. Antibodies that specifically bind to KIR block the interaction between killer cell immunoglobulin-like receptors (KIR) on NK cells and their ligands. Blocking these receptors aids activation of NK cells and it is possible to destroy tumor cells by NK cells. Examples of anti-KIR antibodies have been disclosed in international publication nos. WO/2014/055648, WO 2005/003168, WO 2005/009465, WO 2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/065939, WO 2012/071411 and WO/2012/160448.

In some aspects, an anti-KIR antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-KIR antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-KIR antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-KIR antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-KIR antibody; and (h) about 2000U/mL rHuPH20.

One anti-KIR antibody useful in the present disclosure is the Li Ruilu mab first described in international publication No. WO 2008/084106 (also referred to as the S241P variant of BMS-986015, IPH2102, or 1-7F 9). Additional anti-KIR antibodies useful in the present disclosure are 1-7F9 (also known as IPH 2101) described in International publication No. WO 2006/003179. In one aspect, an anti-KIR antibody for use in a composition of the invention cross competes with Li Ruilu mab or I-7F9 for binding to KIR. In another aspect, the anti-KIR antibody binds to the same epitope as Li Ruilu mab or I-7F 9. In other aspects, the anti-KIR antibody comprises six CDRs of Li Ruilu mab or I-7F 9.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) KIR and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) KIR and (ii) CD3.

III.G.5. anti-GITR antibodies

In some aspects, the second antibody comprises an anti-GITR antibody. anti-GITR antibodies include any anti-GITR antibody that specifically binds to a human GITR target and activates glucocorticoid-induced tumor necrosis factor receptor (GITR). GITR is a member of the TNF receptor superfamily that is expressed on the surface of multiple types of immune cells, including regulatory T cells, effector T cells, B cells, natural Killer (NK) cells, and activated dendritic cells ("anti-GITR agonist antibodies"). Specifically, GITR activation increases proliferation and function of effector T cells, as well as abrogates inhibition induced by activated T regulatory cells. In addition, GITR stimulation promotes anti-tumor immunity by increasing the activity of other immune cells (e.g., NK cells, antigen presenting cells, and B cells). Examples of anti-GITR antibodies have been disclosed in international publication nos. WO/2015/031667, WO 2015/184,099, WO 2015/026,684, WO 11/028683 and WO/2006/105021, U.S. patent nos. 7,812,135 and 8,388,967, and U.S. publication nos. 2009/0132694, 2014/0220002, 2013/0183321 and 2014/0348841.

In some aspects, an anti-GITR antibody can be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-GITR antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-GITR antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-GITR antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-GITR antibody; and (h) about 2000U/mL rHuPH20.

In one aspect, an anti-GITR antibody useful in the present disclosure is TRX518 (described, for example, in Schaer et al Curr Opin immunol. (2012) for 4 months; 24 (2): 217-224 and WO/2006/105021). In another aspect, the anti-GITR antibody is selected from MK4166, MK1248, and antibodies described in WO 11/028683 and U.S.8,709,424 and comprising, for example, a VH chain comprising SEQ ID NO 104 and a VL chain comprising SEQ ID NO 105 (wherein the SEQ ID NO is from WO 11/028683 or U.S.8,709,424). In certain aspects, the anti-GITR antibody is an anti-GITR antibody disclosed in WO 2015/031667, e.g., an antibody comprising VH CDRs 1-3 comprising SEQ ID NOS 31, 71 and 63 of WO 2015/031667, and VL CDRs 1-3 comprising SEQ ID NOS 5, 14 and 30 of WO 2015/031667, respectively. In certain aspects, the anti-GITR antibody is an anti-GITR antibody disclosed in WO 2015/184099, e.g., antibodies Hum231#1 or Hum231#2, or CDRs thereof, or derivatives thereof (e.g., pab1967, pab1975, or pab 1979). In certain aspects, the anti-GITR antibody is an anti-GITR antibody disclosed in JP 2008278814, WO 09/009116, WO 2013/039954, US 20140072566, US 20140072565, US 20140065152 or WO 2015/026684, or is INBRX-110 (INHIBRx), LKZ-145 (Novartis) or MEDI-1873 (MedImmune). In certain aspects, the anti-GITR antibody is an anti-GITR antibody described in PCT/US 2015/033991 (e.g., an antibody comprising the variable region of 28F3, 18E10, or 19D 3).

In certain aspects, the anti-GITR antibodies cross-compete with anti-GITR antibodies described herein (e.g., TRX518, MK4166, or antibodies comprising the VH domain and VL domain amino acid sequences described herein). In some aspects, the anti-GITR antibody binds to the same epitope as an anti-GITR antibody described herein (e.g., TRX518, MK4166, or an antibody comprising the VH domain and VL domain amino acid sequences described herein). In certain aspects, the anti-GITR antibodies comprise six CDRs of TRX518, MK4166, or those of an antibody comprising the VH domain and VL domain amino acid sequences described herein.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) GITR and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) GITR and (ii) CD3.

III.G.6. anti-TIM 3 antibodies

In some aspects, the second antibody comprises an anti-TIM 3 antibody. In some aspects, the anti-TIM 3 antibody comprises an anti-TIM 3 antibody selected from the group consisting of: international publication Nos. WO 2018013818, WO/2015/11702 (e.g., MGB453, novartis), WO/2016/161270 (e.g., TSR-022, tesaro/AnaptyrsBio), WO 2011155607, WO 2016/144803 (e.g., STI-600,Sorrento Therapeutics), WO 2016/071448, WO 17055399; WO 17055404, WO 17178493, WO 18036561, WO 18039020 (e.g. Ly-3221367,Eli Lilly), WO 2017205721, WO 17079112; WO 17079115; WO 17079116, WO 11159877, WO 13006490, WO 2016068802, WO 2016068803, WO 2016/111947, WO/2017/031242.

In some aspects, an anti-TIM 3 antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-TIM 3 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-TIM 3 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-TIM 3 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-TIM 3 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) TIM-3 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) TIM-3 and (ii) CD3.

III.G.7. anti-OX 40 antibodies

In some aspects, the second antibody comprises an anti-OX 40 (also known as CD134, TNFRSF4, ACT35, and/or TXGP 1L) antibody. In some aspects, the anti-OX 40 antibody includes BMS-986178 (Bristol-Myers Squibb Company) described in International publication No. WO 20160196228. In some aspects, the anti-OX 40 antibody comprises an anti-OX 40 antibody selected from the group consisting of those described in the following documents: international publication Nos. WO 95012673, WO 199942585, WO 14148895, WO 15153513, WO 15153514, WO 13038191, WO 16057667, WO 03106498, WO 12027328, WO 13028231, WO 16200836, WO 17063162, WO 17134292, WO 17096179, WO 17096281 and WO 17096182.

In some aspects, the anti-OX 40 antibody may be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-OX 40 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-OX 40 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-OX 40 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-OX 40 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) OX40 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) OX40 and (ii) CD3.

III.G.8. anti-NKG 2A antibodies

In some aspects, the second antibody comprises an anti-NKG 2A antibody. NKG2A is a member of the C-type lectin receptor family, which is expressed on Natural Killer (NK) cells and a subset of T lymphocytes. Specifically, NKG2A is expressed primarily on tumor-infiltrating innate immune effector NK cells as well as on some cd8+ T cells. Its natural ligand human leukocyte antigen E (HLA-E) is expressed on solid tumors and blood tumors. NKG2A is an inhibitory receptor that binds HLA-E.

In some aspects, an anti-NKG 2A antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-NKG 2A antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-NKG 2A antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-NKG 2A antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-NKG 2A antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the anti-NKG 2A antibody comprises BMS-986315, a human monoclonal antibody that blocks the interaction of NKG2A with its ligand HLA-E, thereby allowing activation of an anti-tumor immune response. In some aspects, the anti-NKG 2A antibody comprises a checkpoint inhibitor that activates T cells, NK cells, and/or tumor-infiltrating immune cells. In some aspects, the anti-NKG 2A antibody comprises an anti-NKG 2A antibody selected from those described in, for example, the following documents: WO 2006/070286 (Innate Pharma S.A.; university of Genova); U.S. patent No. 8,993,319 (Innate Pharma S.A.; university of Genova); WO 2007/042573 (Innate Pharma S/A; novo Nordisk A/S; university of Genova); U.S. Pat. No. 9,447,185 (Innate Pharma S/A; novo Nordisk A/S; university of Genova); WO 2008/009545 (Novo Nordisk a/S); U.S. patent No. 8,206,709;8,901,283;9,683,041 (Novo Nordisk A/S); WO 2009/092805 (Novo Nordisk a/S); U.S. Pat. Nos. 8,796,427 and 9,422,368 (Novo Nordisk A/S); WO 2016/134371 (Ohio State Innovation Foundation); WO 2016/03334 (Janssen); WO 2016/04947 (Innate); WO 2016/04945 (Academisch Ziekenhuis Leiden H.O.D.N.LUMC); WO 2016/04947 (Innate Pharma); and WO 2016/04945 (Innate Pharma).

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) NKG2A and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) NKG2A and (ii) CD3.

III.G.9. Anti-ICOS antibodies

In some aspects, the second antibody comprises an anti-ICOS antibody. ICOS is an immune checkpoint protein, a member of the CD28 superfamily. ICOS is a 55-60kDa type I transmembrane protein that is expressed on T cells following T cell activation, and co-stimulates T cell activation following binding to its ligand ICOS-L (B7H 2). ICOS is also known as inducible T cell costimulatory, CVID1, AILIM, inducible costimulatory, CD278, activation-induced lymphocyte immune-mediating molecule and CD278 antigen.

In some aspects, the anti-ICOS antibody can be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-ICOS antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-ICOS antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-ICOS antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-ICOS antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the anti-ICOS antibody comprises BMS-986226, a humanized IgG monoclonal antibody that binds to and stimulates human ICOS. In some aspects, the anti-ICOS antibody comprises an anti-ICOS antibody selected from the group consisting of those described in, for example, the following documents: WO 2016/154177 (Jounce Therapeutics, inc.), WO 2008/137915 (medimune), WO 2012/131004 (INSERM, french National Institute of Health and Medical Research), EP3147297 (INSERM, french National Institute of Health and Medical Research), WO 2011/04613 (Memorial Sloan Kettering Cancer Center), EP 2482849 (Memorial Sloan Kettering Cancer Center), WO 1999/15553 (Robert Koch Institute), U.S. patent No. 7,259,247and 7,722,872 (Robert Kotch Institute); WO 1998/038216 (Japan Tobacco inc.), U.S. patent No. 7,045,615;7,112,655 and 8,389,690 (Japan Tobacco inc.), U.S. Pat. nos. 9,738,718 and 9,771,424 (GlaxoSmithKline) and WO 2017/220988 (Kymab Limited).

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) ICOS and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) ICOS and (ii) CD3.

III.G.10. anti-TIGIT antibodies

In some aspects, the second antibody comprises an anti-TIGIT antibody. In some aspects, the anti-TIGIT antibody comprises BMS-986207. In some aspects, the anti-TIGIT antibody comprises clone 22G2, as described in WO 2016/106302. In some aspects, the anti-TIGIT antibodies comprise MTIG7192A/RG6058/RO7092284 or clone 4.1D3, as described in WO 2017/053748. In some aspects, the anti-TIGIT antibody comprises an anti-TIGIT antibody selected from those described in, for example, the following documents: WO 2016/106302 (Bristol-Myers Squibb Company) and WO 2017/053748 (Genntech).

In some aspects, an anti-TIGIT antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-TIGIT antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-TIGIT antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-TIM 3 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-TIGIT antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) TIGIT and (ii) a second antigen. In some aspects, the antibody comprises a TIGIT bispecific antibody that specifically binds to (i) TIGIT; and (ii) an inhibitory receptor expressed on T cells, NK cells, or both T cells and NK cells. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) TIGIT and (ii) CD3.III.G.11. anti-IL-12 antibodies

In some aspects, the second antibody comprises an anti-IL-12 antibody. In certain aspects, the pharmaceutical composition comprises: (a) anti IL-12 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) anti IL-12 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the anti-IL-12 antibody can be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) anti-IL-12 antibodies. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) anti-IL-12 antibodies; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) anti-IL-12 antibodies. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) anti-IL-12 antibodies; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) IL-12 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) IL-12 and (ii) CD3.

III.G.12. anti-IL-13 antibodies

In some aspects, the second antibody comprises an anti-IL-13 antibody. In certain aspects, the pharmaceutical composition comprises: (a) anti-IL-13 antibodies; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) anti-IL-13 antibodies; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, an anti-IL-13 antibody can be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-IL-13 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-IL-13 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-IL-13 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-IL-13 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) IL-13 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) IL-13 and (ii) CD3.

III.G.13. anti-IL-15 antibodies

In some aspects, the second antibody comprises an anti-IL-15 antibody. In certain aspects, the pharmaceutical composition comprises: (a) anti-IL-15 antibodies; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) anti-IL-15 antibodies; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, an anti-IL-15 antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-IL-15 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-IL-15 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-IL-15 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-IL-15 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) IL-15 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) IL-15 and (ii) CD3.

III.G.14. anti SIRP alpha antibodies

In some aspects, the second antibody comprises an anti-sirpa antibody. In certain aspects, the pharmaceutical composition comprises: (a) an anti-sirpa antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) an anti-sirpa antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, an anti-sirpa antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-sirpa antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-sirpa antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-sirpa antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-sirpa antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) sirpa and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) sirpa and (ii) CD3.

III.G.15. anti-CD 47 antibodies

In some aspects, the second antibody comprises an anti-CD 47 antibody. In certain aspects, the pharmaceutical composition comprises: (a) an anti-CD 47 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) an anti-CD 47 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, an anti-CD 47 antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CD 47 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CD 47 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CD 47 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CD 47 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CD47 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CD47 and (ii) CD3.

III.G.16. Anti-CCR 8 antibodies

In some aspects, the second antibody comprises an anti-CCR 8 antibody. In certain aspects, the pharmaceutical composition comprises: (a) an anti-CCR 8 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) an anti-CCR 8 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, an anti-CCR 8 antibody may be formulated with an anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CCR 8 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CCR 8 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-CCR 8 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-CCR 8 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CCR8 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) CCR8 and (ii) CD3.

III.G.17. Anti-MICA antibodies

In some aspects, the second antibody comprises an anti-MICA antibody. In certain aspects, the pharmaceutical composition comprises: (a) an anti-MICA antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) an anti-MICA antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the anti-MICA antibody may be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-MICA antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-MICA antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-MICA antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-MICA antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) MICA and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) MICA and (ii) CD3.

III.G.18. anti-ILT 4 antibodies

In some aspects, the second antibody comprises an anti-ILT 4 antibody. In certain aspects, the pharmaceutical composition comprises: (a) an anti-ILT 4 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; and (f) about 5mM methionine. In certain aspects, the pharmaceutical composition comprises: (a) an anti-ILT 4 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) about 2000U/mL rHuPH20.

In some aspects, the anti-ILT 4 antibody may be formulated with the anti-PD-1 antibody in any of the formulations disclosed herein as a single formulation. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-ILT 4 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of the anti-PD-1 antibody; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-ILT 4 antibody; and (h) about 2000U/mL rHuPH20.

In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; and (g) an anti-ILT 4 antibody. In certain aspects, the pharmaceutical composition comprises: (a) about 150mg/mL of nivolumab; (b) about 20mM histidine; (c) about 250mM sucrose; (d) about 0.05% w/v polysorbate 80; (e) about 50 μm pentetic acid; (f) about 5mM methionine; (g) an anti-ILT 4 antibody; and (h) about 2000U/mL rHuPH20.

In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) ILT4 and (ii) a second antigen. In some aspects, the antibody is a multispecific antibody, e.g., a bispecific antibody, that specifically (i) ILT4 and (ii) CD3.

III.G.19. additional anticancer agents

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-IL-10 antibody. In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) a long-acting IL-10 molecule. In some aspects, the long-acting IL-10 molecules include IL-10-Fc fusion molecules. In some aspects, the long acting IL-10 molecules include pegylated IL-10, such as AM0010 (ARMO Biosciences).

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-IL-2 antibody. In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) a long-acting IL-2 molecule. In some aspects, the long acting IL-2 includes pegylated IL-2, such as NKTR-214 (Nektar; see U.S. Pat. No. 3,182,06,5086 and WO 15/125159).

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-VISTA antibody.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-CD 96 antibody.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-IL-8 antibody, e.g., having-IL8。

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-tgfβ antibody.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-B7-H4 antibody. In certain aspects, the anti-B7-H4 antibody is an anti-B7-H4 antibody disclosed in international publication No. WO/2009/073533.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-Fas ligand antibody. In certain aspects, the anti-Fas ligand antibody is an anti-Fas ligand disclosed in international publication No. WO/2009/073533.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-CXCR 4 antibody. In certain aspects, the anti-CXCR 4 antibody is an anti-CXCR 4 (e.g., wu Luolu mab (BMS-936564)) disclosed in U.S. publication No. 2014/032208.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-mesothelin antibody. In certain aspects, the anti-mesothelin antibody is an anti-mesothelin disclosed in U.S. patent No. 8,399,623.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-HER 2 antibody. In certain aspects, the anti-HER 2 antibody is Herceptin (U.S. Pat. No. 5,821,337), trastuzumab, or enmetrastuzumab (ado-trastuzumab emtansine) (Kadcyla, e.g., WO/2001/000244).

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-CD 27 antibody. In some aspects, the anti-CD-27 antibody is valdecomab (also known as "CDX-1127" and "1F 5"), which is a human IgG1 antibody that is an agonist of human CD27, as disclosed, for example, in U.S. patent No. 9,169,325.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-CD 73 antibody. In certain aspects, the anti-CD 73 antibody is cd73.4.igg2c219s.igg1.1f.

In some aspects, the pharmaceutical compositions disclosed herein comprise (i) an anti-PD-1 or anti-PD-L1 antibody and (ii) an anti-MICA/B antibody. In certain aspects, the anti-MICA/B antibody is any antibody or antigen binding portion thereof that specifically binds human MICA/B, including, but not limited to, any anti-MICA/B antibody disclosed in international publication No. WO 2019/183551, which is incorporated herein by reference in its entirety.

All references cited above and all references cited herein are incorporated by reference in their entirety.

The following examples are provided by way of illustration and not by way of limitation.

Examples

EXAMPLE 1 subcutaneous formulation development

This example discusses the development of stable, robust Subcutaneous (SC) formulations of nivolumab and manufacturing processes suitable for commercial scale production. As part of formulation studies, the effect of various pharmaceutically acceptable excipients on the stability of nivolumab was evaluated. Studies were also conducted to select processing and packaging components that are compatible with the selected formulation. In addition, time of use studies were conducted to support administration of pharmaceutical products via subcutaneous injection.

Targets for these studies conducted for the development of the nivolumab SC injection include: 1. identification and development of stable injectable formulations for nivolumab SC injection that would be suitable for clinical use and final commercialization; 2. identification of manufacturing equipment and packaging components compatible with the nivolumab SC injection; 3. development and optimization of processes for manufacturing pharmaceutical products; 4. manufacture of three batches of nivolumab SC injections for long-term stability studies and phase 3 clinical trials; transferring the technology for product manufacturing to a commercial production facility and manufacturing PPQ batches.

Formulation development

Buffer system and pH selection

Previous studies related to Intravenous (IV) formulation development of nivolumab evaluated protein stability as a function of solution pH using capillary Differential Scanning Calorimetry (DSC), which measures the thermodynamics of protein unfolding; and further evaluate the physical stability of appropriate nivolumab in buffer at pH 6.0 and 7.0 for formulation. Based on the results of the two studies, 20mM citrate buffer at pH 6.0 was selected for the IV formulation of nivolumab.

Although citrate has proven to be a suitable buffer for IV nivolumab drug products, it would not be a preferred buffer for products for subcutaneous administration, as citrate buffers are known to cause stinging and burning pain after SC administration.

In an effort to identify additional buffers suitable for SC formulations at a target pH of 6.0, studies were conducted to examine the stability of high concentrations (100 mg/mL) of nivolumab in 20mM histidine buffer formulations at pH values ranging from 5.5 to 6.5. Stability data for samples stored at 40 ℃ stress conditions are presented in table 2. Although the rate of formation of high molecular weight species is relatively consistent across the pH range, HMWS levels are minimal in the pH 6.0 sample after two months of storage at 40 ℃. It was also observed that the main peak area determined by iCIEF was highest for the sample at pH 6.0. Based on the results of these studies, 20mM histidine buffer at target pH 6.0 was selected for formulation of nivolumab SC injection.

Formulation buffer concentration selection

A study was conducted to evaluate the effect of histidine buffer concentration on solution stability of nivolumab. A solution sample was prepared at a concentration of 100mg/mL of nivolumab at pH 6.0. Histidine concentrations were adjusted to 10mM, 20mM and 30mM. Each formulation also contained 250mM sucrose, 0.05% w/w polysorbate 80 and 50. Mu.M pentetic acid. The sample was filled into 3cc glass vials, which were stored at 25 ℃ under accelerated storage conditions for up to 6 months.

Throughout the study, all samples were clear to pale milky, colorless to pale yellow in appearance. Additional stability results are presented in tables 3 and 4. Of the three histidine concentrations, there was no change in solution pH or protein concentration. The levels of the high and low molecular weight species increased with time at the same approximate rate during storage for 6 months. The level of sub-visible particles was low with no obvious trend.

Table 4 presents data for size variants determined by both reducing and non-reducing CE-SDS. The percentage purity, determined by both reducing and non-reducing CE-SDS, was unchanged during 6 months of storage at 25 ℃ acceleration. The data for the charge variants determined by iCIEF are also presented in table 4. The levels of acidic species for all 3 formulations increased by about 8% over 6 months of storage at 25 ℃. The increase in the acidic species is accompanied by a substantially equal decrease in the main peak area. The level of alkaline species was barely changed during the 6 month storage period. Based on the results of this study, a histidine buffer concentration of 20mM was selected for the formulation of the nivolumab SC injection.

Tension regulator selection

A study was conducted to evaluate the effect of sucrose on the stability of nivolumab in high concentration protein formulations. Solutions were prepared at a concentration of 100mg/mL of nivolumab in 20mM histidine buffer (pH 6.0) with sucrose concentrations ranging from 200mM to 400mM. Samples were monitored for up to 7 months under accelerated conditions at 25 ℃. All samples remained clear to pale milky, colorless to pale yellow in appearance. The results of the study presented in table 5 show no change in formulation pH or nivolumab concentration. All samples had low levels of sub-visible particles with no apparent trend. The rates of formation of the high and low molecular weight species are consistent over the range of sucrose concentrations evaluated in the study.

Based on the results of the stability study, a sucrose concentration of 250mM (85.6 g/L) was selected as a tonicity adjuster for the Nawuzumab SC injection formulation. As presented in fig. 1, both the viscosity and osmotic pressure of the formulation increased with increasing sucrose concentration. However, based on the results in table 5, it was found that an increase in sucrose concentration had little effect on the quality attributes of the formulation.

Selection of surfactant concentration

The use of polysorbate 80, a nonionic surfactant, in formulations was evaluated. Four different concentrations of polysorbate 80 (0.01% w/v, 0.03% w/v, 0.05% w/v and 0.07% w/v) were evaluated for their effect on stability of the nivolumab SC injection along with polysorbate 80 free samples. In the initial screening portion of the study, the formulation was prepared and then subjected to six freeze-thaw cycles (-60 ℃ to 25 ℃). The formulation was also vigorously stirred on a wrist shaker for 60 minutes at room temperature/room illumination. Upon exposure to freeze-thawing or agitation, a plurality of visible particles were observed in the polysorbate 80 free sample. Despite the reduced amounts, visible particles were also observed in the 0.01% w/v polysorbate 80 samples exposed to freeze thawing or agitation. No visible particles were observed in samples containing polysorbate 80 concentrations of 0.03% w/v, 0.05% w/v or 0.07% w/v. Thus, it was concluded that polysorbate 80 concentrations below 0.01% w/v for the nivolumab SC injection were insufficient to prevent formulation of visible particles due to freeze-thawing or stress from vigorous agitation.

In the second part of this study, the effect of polysorbate 80 concentration on stability of the nivolumab SC injection was examined. Samples with polysorbate 80 concentrations of 0.03% w/v, 0.05% w/v and 0.07% w/v were stored under stress conditions at 40 ℃ and stability was monitored for two months. The solution appearance did not change throughout the study period, and as presented in table 6, the pH or protein concentration did not change. The formation rates of the high and low molecular weight species are relatively comparable among the three polysorbate 80 concentrations. All samples had low levels of sub-visible particles with no apparent trend. Based on the results of this study, a target polysorbate 80 concentration of 0.05% w/v was selected for the formulation of the nivolumab SC injection.

Addition of metal ion chelating agents

EDTA and DTPA (pentetic acid) were evaluated as potential metal chelators to be incorporated into formulations for use in Nawuzumab SC injections. When samples containing nivolumab were labeled with metal, a preliminary screening study was performed to compare EDTA and pentetic acid performance. In this study, a solution containing nivolumab at 10mg/mL was prepared in 20mM histidine (pH 6.0) with 260mM sucrose. The samples were labeled with metal to concentrations of 500ppb iron, 15ppb chromium, 15ppb nickel, 30ppb copper, 10ppb molybdenum, and 10ppb manganese. Unlabeled samples were also prepared for control. The following six formulations were prepared: 1. formulation a: no metal is added for marking, and no EDTA or pentetic acid is used; 2. formulation B: no metal is added for marking, 50 mu M pentetic acid; 3. formulation C: no metal labeling, 50 μm EDTA; formulation D: metal is marked, and EDTA or pentetic acid is not used; 5. formulation E: metal labelling, 50 μm pentetic acid; formulation F: the metal was labeled with 50. Mu.M EDTA.

The samples were placed under stress conditions at 40 ℃ and the levels of HMWS and LMWS were monitored for up to 4 weeks. The data presented in table 7 shows the benefits of adding a metal ion chelating agent. For formulation D, the level of HMWS increased from 0.27% to 7.07% and the level of LMWS increased from 0.12% to 0.32% over 4 weeks of storage at 40 ℃ with the samples labeled with metal but without chelating agent. EDTA at a concentration of 50. Mu.M (formulation F) was able to limit the increase in HMWS and LMWS levels to 3.75% and 0.18%, respectively, for the metal-labeled samples. However, the best performance of the metal-labeled samples was observed with 50 μm pentetic acid, where the level of HMWS increased only to 0.55% and the level of LMWS did not change over the 4-cycle study period. Even the untagged formulations A, B and C show the benefit of adding a metal ion chelating agent. Without the addition of chelating agent, the level of HMWS increased from 0.27% to 1.94% and the level of LMWS increased from 0.12% to 0.17% over 4 weeks of storage at 40 ℃. When 50 μm pentetic acid or 50 μm EDTA is included in the formulation, the increase in HMWS decreases, increasing from 0.26% to 0.48% for pentetic acid and from 0.26% to 0.52% for EDTA. LMWS levels were unchanged in samples containing metal ion chelators during the 4-cycle storage period. Based on the results of this study, pentetic acid was selected as the metal ion chelator for the nivolumab SC injection formulation.

To further investigate the benefits of metal ion chelators in preventing degradation that may occur due to the presence of trace metal ions, a second more intensive study was conducted to evaluate the effect of pentetic acid on the stability of the nivolumab SC injection. The formulation tested in this study was 120mg/mL nivolumab in 20mM histidine buffer (pH 6.0) containing 250mM sucrose and 0.05% w/v polysorbate 80. The solution was labeled with a concentrated metal solution such that the total metal concentration in the metal-labeled sample was 1.5ppm (0.5 ppm each of iron, chromium and copper). As in the previous study, unlabeled samples were also prepared for control. The following five formulations were prepared: 1. formulation a: no metal is added for marking, and no pentetic acid is generated; 2. formulation B: no metal is added for marking, 50 mu M pentetic acid; 3. formulation C:1.5ppm metal is marked without pentetic acid; 4. formulation D:1.5ppm metal addition, 50. Mu.M pentetic acid; 5. Formulation E:1.5ppm metal was labeled, 100. Mu.M pentetic acid.

Samples were filled into stoppered 3cc glass vials and stored under stress conditions at 40 ℃ and stability was monitored for two months. The solution appearance did not change throughout the study period, and as presented in table 8, the pH or protein concentration did not change. All samples had low levels of sub-visible particles with no apparent trend. After 2 months of storage at 40 ℃, the levels of HMWS and LMWS were highest in formulation C (metal-plus-standard solution without pentetic acid addition). Lower and equal levels of HMWS and LMWS were observed in formulations D and E containing 50 μm and 100 μm pentetic acid, respectively. The results from the unlabeled formulations a and B also show the benefits of adding pentetic acid. Without the addition of chelating agent (formulation a), the level of HMWS increased from 0.61% to 4.34% and the level of LMWS increased from 0.05% to 0.25% during 2 months of storage at 40 ℃. When 50 μm pentetic acid (formulation B) was included in the formulation, the HMWS level at the 2 month time point was only 2.97%, and the LMWS level at the 2 month time point was only 0.20%. Based on the results of this study, a target pentetic acid concentration of 50 μm was selected for the formulation of the nivolumab SC injection.

Selection of protein concentration

Multiple experiments were performed to help select target protein concentrations for the nivolumab SC injections. The formulation was found to be stable during the run and ultrafiltration run allowed for up to 200mg/mL of nivolumab concentration. However, it was found that at a concentration of nivolumab greater than 150mg/mL, the viscosity increased rapidly.

L-arginine is an amino acid known to reduce the viscosity of solutions. To evaluate the effect of arginine on formulation viscosity, a study was conducted in which 75mM arginine was added to 140mg/mL of a nivolumab SC formulation (in 20mM histidine, 250mM sucrose, 0.05% w/v polysorbate 80, 50 μm pentetic acid, pH 6.0) and then concentrated using a 10kDa membrane and centrifuge. The effect of arginine addition on the viscosity of the formulation is shown in figure 2. At a concentration of nivolumab greater than 100mg/mL, 75mM arginine causes a decrease in solution viscosity. For example, 140mg/mL of the nivolumab formulation had a viscosity of 13.3cP in the absence of arginine at 20℃but 9.1cP with 75mM arginine. The viscosity measurements were carried out at 20 ℃.

A study was then conducted to evaluate the effect of 75mM arginine on the stability of the Nawuzumab SC injection (ELN A0C6F-023, ELN A259D-007). A small batch of nivolumab was prepared in 20mM histidine (pH 6.0) with 250mM sucrose, 0.05% w/v polysorbate and 50. Mu.M pentetic acid. The concentration of nivolumab was 100mg/mL or 140mg/mL, and solutions were prepared with and without 75mM arginine. Typical stability data were generated for the quality attributes monitored, but at the 3 month 40 ℃ time point, a number of small white visible particles were observed in the arginine-containing formulation. No visible particles were observed in the arginine-free formulation. Based on these stability results, it was decided that arginine would not be included in the formulation for the nivolumab SC injection.

The target concentration of nivolumab selected for the nivolumab SC injection was 120mg/mL. The formulation selected for FIH clinical trials was 20mM histidine buffer (pH 6.0) containing 250mM sucrose, 0.05% w/v polysorbate 80 and 50. Mu.M pentetic acid. The drug substance was provided in the same formulation, but at a target concentration of 150mg/mL, stored at-60 ℃.

Stability data for laboratory-scale batches of nivolumab SC injections

Two laboratory-scale batches of nivolumab SC injections were prepared and placed in steady state (placed on stability). Two batches were used with the selected FIH formulation, namely 20mM histidine buffer (pH 6.0), 250mM sucrose, 0.05% w/v polysorbate 80 and 50. Mu.M pentetic acid. To include (brecket) a protein concentration of 120mg/mL was selected for the FIH formulation, with one batch having a concentration of 100mg/mL of nivolumab and another batch having a concentration of 140mg/mL of nivolumab. Formulations were prepared and small aliquots were aseptically filtered into 3cc type I glass vials. The vials were stoppered, sealed and placed on a station at 5 ℃, 25 ℃ and 40 ℃. At the indicated time points, samples were withdrawn from the stability console and tested for appearance, pH, protein concentration, size homogeneity (by SE-HPLC), sub-visible particulate matter (by HIAC), charge variants (by iCIEF) and molecular size variants (by CE-SDS (R & NR)).

All samples were clear to pale milky, colorless to pale yellow solutions in visual appearance at all time points. Additional stability data are provided in tables 9-11. As shown in table 9, no change in pH or protein concentration was observed throughout the study for both 100mg/mL and 140mg/mL samples. The level of HMWS in the 100mg/mL sample increased by 0.56% and the level of HMWS in the 140mg/mL sample increased by 0.73% over 12 months of storage at 5 ℃. For the accelerated 25 ℃ conditions, after 6 months of storage, the level of HMWS in the 100mg/mL sample increased by 0.88% and the level of HMWS in the 140mg/mL sample increased by 1.15%. The levels of HMWS in the 100mg/mL and 140mg/mL samples increased by 3.17% and 3.92%, respectively, in storage for 3 months at 40 ℃. The level of LMWS was hardly changed in the samples stored at 5 ℃ and 25 ℃, but the level of LMWS was increased by about 0.2% in the samples stored at 40 ℃ for 3 months.

Table 10 presents the results for sub-visible particles and charge variants. All samples had low levels of sub-visible particles at all time points and no obvious trend. The level of acidic species increases over time under all storage conditions. In 12 months of storage at 5 ℃, the acidic species of the 100mg/mL sample increased by 6.3%, and the acidic species of the 140mg/mL sample increased by 5.8%. The acidic species of the 100mg/mL sample increased by 26.0% and the acidic species of the 140mg/mL sample increased by 23.8% at 25℃acceleration. The levels of acidic species increased by 44.9% and 43.9% in the 100mg/mL and 140mg/mL samples, respectively, upon storage for 3 months at 40 ℃. The increase in the acidic species is accompanied by a substantially equal decrease in the main peak area. For all samples, there was little change in the level of alkaline species throughout the study period.

Table 11 presents data for size variants determined by both reducing and non-reducing CE-SDS. The percent purity, as determined by both reducing and non-reducing CE-SDS, was unchanged for 100mg/mL and 140mg/mL samples over 12 months of storage at 5 ℃. The percent purity of the 100mg/mL sample, as determined by reducing CE-SDS, was unchanged and the percent purity of the 140mg/mL sample was reduced by 0.2% during 6 months of storage at 25 ℃. The percent purity of the 100mg/mL sample, as determined by reducing CE-SDS, was reduced by 1.0% and the percent purity of the 140mg/mL sample was reduced by 1.4% in storage for 3 months at 40 ℃. For non-reducing CE-SDS, the percentage purity of 100mg/mL samples was reduced by 2.2% over 6 months of storage at 25 ℃. For the 140mg/mL sample, the percent purity reduction was 1.8%. The reduction in percent purity of 100mg/mL and 140mg/mL samples, as determined by non-reducing CE-SDS, was 2.6% and 3.1%, respectively, in storage for 3 months at 40 ℃.

Description and composition of drug products selected for FIH clinical trials

The drug product selected for FIH clinical trials was nivolumab injections, 960 mg/vial (120 mg/mL). It is described as a sterile, non-pyrogenic, transparent to strong milky, colorless to yellow liquid. In some cases, there may be a few microparticles that are consistent in appearance with proteinaceous particles. The pharmaceutical product is a disposable, preservative-free isotonic aqueous solution for Subcutaneous (SC) administration. Packaging Nawuzumab SC injection in 10cc 1 flint glass vial with 20mm Daikyo D21-7S The coated butyl plug was stoppered and secured with a 20mm aluminum seal with flip-off cap. The composition of the nivolumab SC injection is presented in table 12, which includes the quality criteria and function of each component. Overfill of the nivolumab SC injection was included in each vial to ensure that an amount of 8.0mL of label could be administered to the patient. In determining overfill of a pharmaceutical product, consider the following: 1.0.5mL for loss in vials, needles and syringes (VNS) during use of the product (with USP<1151>Minimum recommended excess volume fill is consistent); 2.0.2ml for closing losses in the system transfer device (if used); 3.0.5mL for airfoil infusionStartup loss in the accumulator (if used); and 4.0.3ml for filling machine variability.

Based on the amount of the nivolumab SC injection that may be lost during dose preparation and administration, 1.5mL of overfill was included in each vial of drug product.

^a The target fill included 1.5mL overfill to compensate for vial, needle and syringe (VNS) hold-up, filling machine variability, and administration component hold-up

USP = united states pharmacopeia, ph. Eur = european pharmacopeia, NF = national formulary, JP = japanese pharmacopeia, q.s. = IND sufficient to allow stable batches of FIH formulation

A development laboratory batch of nivolumab SC injection (FIH formulation) was manufactured and placed in steady state. The batch size was 3,000ml in size and it resulted in 291 vials after inspection. The drug product was filled (target fill 9.5 mL) into Schott 10cc type I flint glass vials, which were closed with 20mm Daikyo D-21-7-S Flurotec coated butyl rubber stoppers. The vials were sealed with 20mm West aluminum seals with flip-off caps. Vials of the drug product were placed on the operating table at 5 ℃, 25 ℃ and 40 ℃. At the indicated time point, samples were withdrawn from the stability console and tested. Stability data for twelve months are presented in tables 13-16.

All samples were clear to pale milky, colorless to pale yellow solutions in visual appearance at all time points. Additional stability data are provided in tables 13-16. As shown in table 13, no change in pH or protein concentration was observed throughout the study. The level of HMWS increases by 0.3% over 12 months of storage at 5 ℃. After 6 months of storage at 25℃acceleration, the level of HMWS increased by 0.6%. The level of HMWS increases by 3.3% during storage for 3 months at 40℃under stress. The level of LMWS was hardly changed in the samples stored at 5 ℃ and 25 ℃, but the level of LMWS was increased by 1.1% in the samples stored at 40 ℃ for 3 months.

^a Compliance = clear to strong milky white, colorless to yellow liquid, light (very few) particles (consistent in appearance with protein particles) may be present.

Table 14 presents data for size variants determined by both reducing and non-reducing CE-SDS. The percentage purity, determined by both reducing and non-reducing CE-SDS, was unchanged during 12 months of storage at 5 ℃. The percent purity, as determined by reducing CE-SDS, was reduced by 0.2% during 6 months of storage at 25 ℃ acceleration. The percent purity, as determined by reducing CE-SDS, was reduced by 2.9% during storage for 3 months at 40 ℃ under stress conditions. For non-reducing CE-SDS, the percent purity was reduced by 1.2% over 6 months of storage at 25 ℃, and 4.4% over 3 months of storage under stress conditions at 40 ℃.

The level of acidic species increases over time under all storage conditions. The acidic species increased by 0.8% during 12 months of storage at 5 ℃. The acidic species increased by 11.3% during 6 months of storage at 25 ℃. The level of acidic species increased by 38.8% during storage for 3 months at 40 ℃. The level of alkaline species was hardly changed throughout 12 months of storage at 5 ℃. The level of alkaline species increased by 1.6% in 6 months at 25 ℃, and by 2.6% after 3 months of storage at 40 ℃. The observed increase in the acidic species and basic species was accompanied by a substantially equal decrease in the main peak area.

Table 16 presents the results of the active binding ELISA, cell-based bioassays, and sub-visible microparticles. The range of results for the active binding ELISA was 95% to 112% and the range of results for the cell-based bioassays was 79% to 105% at all temperatures and time points. All samples had low levels of sub-visible particles at all time points and no obvious trend.

Clinical batch manufacture (FIH formulation)

Two clinical batches (batch 1 and batch 2) of nivolumab SC injections were made using the FIH formulation. The final batch size of the two batches was about 20 liters, which was filled into about 1,800 vials. Both batches passed all final tests and were released for clinical use. The release test results for these first two clinical batches of nivolumab SC injections are presented in table 17.

A simple description of the manufacturing process is provided below. 150mg/mL of the nivolumab drug substance in 20mM histidine, 250mM sucrose, 0.05% (w/v) polysorbate 80 and 50. Mu.M pentetic acid (pH 6.0) was thawed at room temperature, protected from light, and there was sufficient space between the containers to ensure thawing efficiency. Once the drug substance is completely thawed, the bag is manually mixed for 2 to 3 minutes to ensure homogeneity. A formulation buffer solution (20 mM histidine, 250mM sucrose, 0.05% (w/v) polysorbate 80 and 50. Mu.M pentetic acid, pH 6.0) was prepared and then filtered through a 0.22 μm filter. A specific amount of buffer solution was added to the drug substance to adjust the protein concentration to 120mg/mL. Samples were taken for protein concentration determination, pH and endotoxin testing. 120mg/mL of the drug product solution was filtered through a 0.45 μm prefilter. Samples were taken for bioburden testing. The drug product solution was filtered through two 0.22 μm filters. The filters were subjected to pre-filtration and post-filtration integrity tests. The sterile filtered solution is then filled into washed, sterilized, depyrogenated vials. The vials were stoppered with sterile stoppers and sealed with aluminum seals. During the filling process, a filling check by weight is performed periodically. The sealed vials were visually inspected for defects at 100%. The inspected vials are labeled and packaged.

Formal use-time/compatibility study

A use-time study was performed to support subcutaneous clinical administration of nivolumab injections, 960 mg/vial (120 mg/mL). In the initial clinical study, nivolumab (SC) was administered subcutaneously to patients using one of two methods. For method 1, after adding small aliquots of saline (NS) and rHuPH20 (ENHANZE drug product (EDP)) to adjust the concentration of nivolumab in the vial to 109.1mg/mL and rHuPH20 to 2,000u/mL, nivolumab was administered as bolus subcutaneous injections at doses of 480mg, 720mg and 960mg at rates between 2 and 4 milliliters per minute. At a concentration of 109.1mg/mL, SC administration volumes of 4.4mL, 6.6mL, and 8.8mL provided doses of 480mg, 720mg, and 960mg, respectively, of nivolumab. For method 2, 960mg (8 mL of 120 mg/mL) was administered over about 30 minutes (about 0.27 mL/min) using a syringe pump (without addition of NS and EDP). To simulate the worst case in this use-time study, a solution flow rate of up to 4 ml/min was defined as it passed through a 27g 1/2 "needle. Furthermore, once the drug product is in the administration syringe, a holding period of up to 24 hours is defined at 2-8 ℃, of which 24 hours there is room temperature/room illumination (RT/RL).

For method 1, 0.76mL NS and 0.19mL EDP were added to the vial of nivolumab SC injection, which was then gently swirled and inverted for mixing. The vial contents were drawn into a separate 10cc syringe. At the initial time point, the vial contents were expelled into the sampling vessel via a wing-type infusion set (27 g 1/2 "needle) at a rate of 4 mL/min. The tip cap was applied to another set of filled syringes, which were kept at RT/RL for 4 hours, then at 2 ℃ -8 ℃ protected from light, and kept for an additional 20 hours. At the 24 hour time point, the vial contents were again expelled into the sampling vessel via the airfoil infusion set at a rate of 4 mL/min. For method 2, the vial contents (without NS or EDP added) were drawn into a separate 10cc syringe. Samples were collected, stored and sampled as described above for method 1.

Results from the use-time study are presented in tables 18-21. Table 18 shows the results of protein concentration and size homogeneity as determined by SE-HPLC. There was no change in protein concentration or level of high or low molecular weight species throughout the study period.

Table 19 presents the results of the test for solution appearance, pH, binding activity, and sub-visible particulate matter. There was little change in both solution pH and active binding throughout the study period. The level of sub-visible particles in the sample was low and there was no apparent trend.

^a The light yellow, concentrated milky white solution, as indicated, was substantially free of visible particles.

Table 20 shows the data for the percent purity and the sum of all minor peaks (. Gtoreq.0.3%) as determined by CE-SDS.

The results show that the level of size variants was unchanged throughout the study period for all samples.

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Table 21 presents the enzyme activity and data for determining charge variants by iCIEF. The results showed that the enzyme activity was relatively unchanged throughout the study period. The levels of acidic species, main peak and basic species were unchanged during the 24 hour study period.

Based on the results of this usage-time study, the following conclusions were drawn: 1. the nivolumab SC injection was stable when diluted to a protein concentration of 109mg/mL with 0.76mL of 0.9% sodium chloride injection (NS) and 0.19mL of ENHANZE Drug Product (EDP) and stored in a plastic syringe at 2 ℃ -8 ℃ for up to 24 hours (4 hours of said 24 hours at room temperature and indoor illumination); 2. 120mg/mL of nivolumab SC injection was stable when stored in plastic syringe at 2 ℃ -8 ℃ for up to 24 hours (4 hours of said 24 hours at room temperature and indoor illumination); the Nawuzumab SC injection and the 120mg/mL Nawuzumab SC injection diluted with NS and EDP to protein concentrations of 109mg/mL were compatible with PVC administration set and were capable of passing through syringe needles at flow rates up to 4 mL/min.

Commercial formulation development

In the initial clinical trial discussed above, rHuPH20 enzyme was added to vials of nivolumab SC injections immediately prior to subcutaneous administration of the dose to the patient. This study evaluates that the addition of rHuPH20 to the formulation allows the dose to be prepared from a single vial of commercial formulation of the nivolumab SC injection.

In the initial clinical trial, theThe drug product (EDP, halozyme Therapeutics) was added to and mixed with nivolumab before being withdrawn into the syringe. EDP is a sterile, non-pyrogenic, disposable, preservative-free isotonic aqueous solution. EDP provides 1mg/mL rHuPH20 in a formulation containing 10mM histidine (pH 6.5), 130mM sodium chloride, 10mM methionine, and 0.02% w/w polysorbate 80. Packaging EDP in 2cc type I fireThe vials were stoppered with chlorinated butyl rubber stoppers and sealed with aluminum seals.

For co-formulated drug products, rHuPH20 drug substances were used in the formulation instead of EDP. The drug substance provided a higher rHuPH20 concentration of 10mg/mL and was formulated with 10mM histidine and 130mM sodium chloride (pH 6.5). The rHuPH20 drug substance was supplied as a frozen solution in a bottle, thawed and gently mixed prior to use. As with the FIH clinical study, the enzyme concentration in the commercial coformulated drug product was maintained at 2,000U/mL.

Initial feasibility study

Initial feasibility studies were performed to evaluate the stability of 100mg/mL nivolumab in the presence of 2,000u/mL rHuPH20 at 5 ℃, 25 ℃ and 40 ℃ when packaged in glass vials. Formulations were prepared with and without 2,000U/mL rHuPH20, filled into 3cc glass vials, stoppered, sealed and placed on a bench. Samples were tested at the initial time points for solution appearance, pH, protein concentration, size homogeneity (by SE-HPLC) and sub-visible particulate matter (by HIAC) and retested at 1 week, 2 weeks and 4 weeks. In addition, charge variants were determined by iCIEF, molecular weight distribution was determined by CE-SDS (R & NR), and enzyme activity was determined using plate-based turbidimetry.

The results generated in this study presented in tables 22-26 show that the presence of 2,000u/mL rHuPH20 in the formulation had no effect on the quality attributes of nivolumab. There was no difference in solution appearance, pH, protein concentration or sub-visible particle levels during 4 weeks of storage at 5 ℃, 25 ℃ and 40 ℃ when comparing the results for the enzyme-containing formulations with the results for the no enzyme control. No differences were observed for the results of size homogeneity (by SE-HPLC), charge variants (by iCIEF) or molecular weight variants (by CE-SDS (R & NR)). Comparable results for enzyme activity were observed at 5 ℃ and 25 ℃, but due to the relatively low Tm of the enzyme, the activity rapidly decreased when stored at 40 ℃.

^a Transparent to pale milky white, colorless to pale yellow solution

^a Inapplicable, b undetected

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Development of commercial formulation-addition of rHuPH20

The main goal in the development of commercial formulations for the nivolumab SC injection was to add rHuPH20 to the FIH clinical formulation. As with FIH administration, the target enzyme activity in the commercial drug product was 2,000 units/ml. The concentration of target protein of the rHuPH20 drug substance was 10mg/mL, the target enzyme activity was 110,000 units/mg, and the density was 1.010g/mL at 20 ℃. Thus, for an enzyme activity of 2,000 units/mL, the theoretical amount of rHuPH20 drug substance required per liter of finished drug product is: DP of 2,000kU/L x 1mg rHuPH20/110kU x 1mL/10mg x 1.010g/mL=1.84 g/L.

1.84g rHuPH20 drug substance/liter drug product resulted in a drug product enzyme concentration of 0.0182mg/mL from: 1.84g/L x 1mL/1.010g x 10mg/mL x 1L/1,000mL = 0.0182mg rHuPh20/mL pharmaceutical product.

The actual amount of rHuPH20 drug substance added during manufacture of the drug product is determined based on the protein concentration and enzyme activity of the rHuPH20 drug substance and may range from 8.5 to 12.5mg/mL and 80 to 140kU/mg, respectively.

Addition of methionine as a sacrificial antioxidant

To prevent oxidation of rHuPH20, the following study was performed in which methionine was added to the formulation as a sacrificial antioxidant. To stress the formulation, peroxide was added to the formulation containing methionine at concentrations of 0mM, 5mM and 10mM at a concentration of 1 mM. A level of 1mM peroxide was selected based on information in the literature stating that about 0.15mM peroxide can be formed in a solution containing 0.05% w/v polysorbate 80 when stored at 40 ℃ for up to 5 weeks.

In this experiment, solutions were prepared at a concentration of 120mg/mL of nivolumab in 20mM histidine buffer (pH 6.0) with 250mM sucrose, 0.05% w/v polysorbate 80, 50. Mu.M pentetic acid, and 2,000U/mL rHuPH 20. The peroxide and methionine concentrations in each solution were adjusted as follows: 1. formulation a: peroxide-free, methionine-free; 2. formulation B: peroxide-free, 10mM methionine; 3. formulation C:1mM peroxide, methionine free; 4. formulation D:1mM peroxide, 5mM methionine; 5. Formulation E:1mM peroxide, 10mM methionine.

Samples were placed on the bench under accelerated conditions at 25 ℃ and monitored for up to 6 months. All samples remained clear to pale milky, colorless to pale yellow in appearance. Additional stability results are presented in table 27. The data show that essentially no differences in formulation pH, nivolumab concentration, or levels of high and low molecular weight species were observed. In addition, the level of sub-visible particles was low for all samples with no obvious trend. One difference observed was that formulation C, which contained peroxide but no methionine, had significantly lower enzyme activity than the other 4 formulations. Even with 1mM peroxide in the formulation, both formulations D and E fully retain enzyme activity. Based on the results of this stability study, a methionine concentration of 5mM was selected for use in commercial pharmaceutical products.

Stability of the Nawuzumab SC injection when formulations were labeled with 1.5ppm metal-evaluation of protective effects of pentetic acid and methionine

As previously discussed, 50 μΜ pentetic acid was selected as a component of the formulation to prevent trace metal catalyzed nivolumab degradation, and 5mM methionine was selected as a sacrificial antioxidant to prevent peroxide induced rHuPH20 oxidation. Studies were conducted to evaluate the effect on stability of the nivolumab SC injection when both excipients were included in the formulation. In this experiment, solutions were prepared at a concentration of 120mg/mL of nivolumab in 20mM histidine buffer (pH 6.0) containing 250mM sucrose and 0.05% w/v polysorbate 80. The pentetic acid and methionine concentrations in each formulation were adjusted as follows: 1. formulation a: no pentetic acid, no methionine; 2. formulation B:50 μm pentetic acid, methionine free; 3. formulation C: 5mM methionine without pentetic acid; formulation D: 50. Mu.M pentetic acid, 5mM methionine.

Each formulation was labeled with a concentrated metal solution such that the final formulation concentration of the metal was 1.5ppm (0.5 ppm each of iron, chromium and copper). Samples of each formulation were filled into vials, placed on a bench under stress conditions at 40 ℃ and monitored for up to 2 months. All samples remained clear to pale milky, colorless to pale yellow in appearance. The additional stability results presented in table 28 show that there was no difference in formulation pH, nivolumab concentration, or levels of sub-visible microparticles. After 2 months of storage at 40 ℃, the level of high molecular weight species was highest in formulation a containing neither pentetic acid nor methionine, and lowest in formulation D containing both excipients. Comparison of HMWS levels in formulation B and formulation C showed that 50 μm pentetic acid formed more protective against HMWS than 5mM methionine. Thus, the results of this study support the inclusion of both 50 μm pentetic acid and 5mM methionine in commercial formulations for the nivolumab SC injection.

IND allowing stability batches of commercial formulations

Laboratory batches of 120mg/mL nivolumab SC injections (commercial formulations) were made and placed in steady state. The formulation was 120mg/mL of nivolumab in 20mM histidine buffer (pH 6.0) with 250mM sucrose, 0.05% w/v polysorbate 80, 50. Mu.M pentetic acid, 5mM methionine, and 2,000 units/mL rHuPH 20. The batch size was 3,000mL in size and it produced 368 vials after injection (800 mL from this batch for other development activities). The drug product was filled (target fill 5.67mL, label strength 600 mg/vial) into a Schott 10R I flint glass vial, which was closed with a 20mm Daikyo D-21-7-S flumotec coated butyl rubber stopper. The vials were sealed with 20mm West aluminum seals with flip-off caps. Vials of the drug product were placed on the operating table at 5 ℃, 25 ℃ and 40 ℃. At the indicated time point, samples were withdrawn from the stability console and tested. IND stability data for twelve months are presented in tables 29 to 32.

All samples met the specification in visual appearance at all time points (clear to strong milky, colorless to yellow liquid, light (few) particles may be present (appearance consistent with protein particles)). Additional stability data are provided in tables 29-32. As shown in table 29, no changes in pH or protein concentration were observed throughout the study. The level of HMWS increased by 1.x% over 12 months of storage at 5 ℃. After 6 months of storage at 25℃acceleration, the level of HMWS increased by 0.6%. The level of HMWS increases by 3.3% during storage for 3 months at 40℃under stress. The level of LMWS was hardly changed in the samples stored at 5 ℃ and 25 ℃, but the level of LMWS was increased by 1.1% in the samples stored at 40 ℃ for 3 months.

Table 30 presents data for size variants determined by both reducing and non-reducing CE-SDS. The percentage purity, determined by both reducing and non-reducing CE-SDS, was unchanged during 12 months of storage at 5 ℃. The percent purity, as determined by reducing CE-SDS, was reduced by 0.2% during 6 months of storage at 25 ℃ acceleration. The percent purity, as determined by reducing CE-SDS, was reduced by 2.9% during storage for 3 months at 40 ℃ under stress conditions. For non-reducing CE-SDS, the percent purity was reduced by 1.2% over 6 months of storage at 25 ℃ and 4.4% under 40 ℃ stress conditions.

The level of acidic species increases over time under all storage conditions. The acidic species increased by 1.5% during 12 months of storage at 5 ℃. The acidic species increased by 11.3% during 6 months of storage at 25 ℃. The level of acidic species increased by 38.8% during storage for 3 months at 40 ℃. The level of alkaline species was hardly changed throughout 12 months of storage at 5 ℃. The level of alkaline species increased by 1.6% in 6 months at 25 ℃, and by 2.6% after 3 months of storage at 40 ℃. The increase in the acidic species and the basic species is accompanied by a substantially equal decrease in the main peak area.

Table 32 presents the results of the active binding ELISA, cell-based bioassays, and sub-visible microparticles. The range of results for the active binding ELISA was 95% to 112% and the range of results for the cell-based bioassays was 79% to 105% at all temperatures and time points. All samples had low levels of sub-visible particles at all time points and no obvious trend.

Formulation robustness

Studies were conducted to examine the robustness of the formulations selected for commercial pharmaceutical products. Study design required 13 different formulations to be prepared. Nine formulations were prepared for primary effect screening, two formulations were prepared with targets, and two axial runs were prepared to determine the effect of protein concentration. Additional vials were prepared for one of the target formulations and stored frozen at-60 ℃. At each time point these frozen samples were thawed and tested to assess the analytical (between time points) differences. Five factors in the formulation prepared varied and included protein concentration (105 mg/mL to 135 mg/mL), pH (5.5 to 6.5), polysorbate 80 concentration (0.025% w/v to 0.075% w/v), pentetic acid concentration (25 μm to 75 μm), and methionine concentration (2.5 mM to 7.5 mM). Formulations prepared for robustness studies are presented in table 33.

The formulation was prepared by first thawing approximately one liter of purified drug substance (150 g/L nivolumab in 20mM histidine, 250mM sucrose (pH 6.0)). The exchange buffer was then filtered using tangential flow, and half of the body was adjusted to pH 5.5 and the other half to pH 6.5. For an intermediate target of pH 6.0, a purified drug substance of pH 5.5 is added to a purified drug substance of pH 6.5 until a target pH of 6.0 is reached. For each of the 14 formulations, a volume of 50mL was prepared. The concentration of polysorbate 80, pentetic acid and methionine in each formulation was adjusted by adding different amounts of concentrated labelling solutions. After preparation, each formulation was passed through a 0.22 μm sterilizing filter, which was then filled into 3cc glass vials, stoppered and sealed. The filled vials were then placed on a bench under recommended 5 ℃ storage conditions and under accelerated stability conditions of 25 ℃.

Each set of samples was tested for solution appearance, pH, protein concentration, size homogeneity (by SE-HPLC) and sub-visible particulate matter (by HIAC) at the initial time points. In addition, charge variants were determined by iCIEF, molecular weight distribution was determined by CE-SDS (R & NR), and enzyme activity was determined using plate-based turbidimetry. The samples were retested after 1 and 3 months of storage at 25 ℃ acceleration and 6 and 12 months of storage at 5 ℃. Stability results are presented in tables 34 to 39.

At the initial time point, there was no change in solution appearance, pH, or nivolumab concentration in all four groups. Similar initial results were observed for size homogeneity (by SE-HPLC), charge variants (by iCIEF), size variants (by CE-SDS (R & NR)) and enzyme activity for groups 1, 3 and 4. The HMWS level of the group 2 samples (room temperature, room illumination and 30 ℃ storage) was 0.21% higher than the control, and the acidic species level of the group 2 samples was 1.4% higher than the control. The sub-visible particulate matter (via HIAC) was very low for all samples. At the initial time point, the enzyme activity of the group 2 samples was 2.5% lower than that of the control.

After six months of storage at 25 ℃ acceleration, there was no change in solution appearance, pH or nivolumab concentration in all four groups. HMWS levels for groups 1, 3 and 4 were similar, and the HWWS level for group 2 was 0.15% higher than the control HWWS level. The LMWS was the same for all four groups, 0.13%. For the charge variant (by iCIEF), the acidic species level of group 2 was 2.7% higher than the acidic species level of the control, and groups 1 and 3 were similar to the control. At six month time points, the range of alkaline species for all four groups was 5.9% to 6.0%. The level of molecular size variants for all four groups was in the range of 99.7% to 99.8% (for reducing CE-SDS) and in the range of 96.5% to 97.2% (for non-reducing CE-SDS). At six month time points, all samples were very low in sub-visible particulate matter counts as determined by HIAC, with no obvious trend, and all groups were determined to have enzyme activity within 99.2% and 105.6% of the control.

^a Compliance = clear to pale milky white, colorless to pale yellow solution

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^a Compliance = clear to pale milky white, colorless to pale yellow solution

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Determination of vial target fill volume and overfill

The labeled SC dose of the nivolumab was 480mg, which is a 4mL injection of a drug product with a nivolumab concentration of 120 mg/mL. Overfilling of the nivolumab SC injection was included in each vial to compensate for loss of vials, needles and syringes (VNS) during use of the product (consistent with USP <1151> minimum recommended overfill) and to compensate for variability in the filling machine. Overfilling ensures that the label-required nivolumab SC injection can be withdrawn from the vial.

A study was conducted to determine the actual nivolumab SC injection retention volume in the vials, needles and syringes. The vials were accurately filled with 4.50mL of the drug product, stoppered and sealed. Five participants drawn the vial contents using a 20g 1.5 "needle using a 5cc plastic syringe. The 20g 1.5 "needle was replaced with a 25g 5/8" needle and the syringe contents were discharged into a small beaker. The weight of the expelled drug product was converted to volume and subtracted from the 4.50mL vial fill volume. The retention volume of 5 participants ranged from 0.29mL to 0.35mL with an average of 0.32mL. Note that this value is similar to the USP <1151> suggested excess of 0.34mL (extrapolated value) for a 6cc vial size.

Closed System Transfer Devices (CSTDs) are used at many facilities to protect healthcare personnel from drug exposure. CSTD for direct SC injection is generally composed of 3 parts and includes a vial adapter, a syringe adapter, and a needle adapter. To determine the hold-up volume in CSTD, the same 5 participants repeated the study with 6 most commonly used CSTDs. Once the CSTD is attached to the vial it cannot be easily removed, and thus this study will determine the hold-up volume in the vial plus the hold-up volumes in the CSTD and syringe.

As in the previous group studied, 6R vials were accurately filled with 4.50mL of nivolumab injectate, then stoppered and sealed. Five participants drawn the vial contents into a 5cc plastic syringe using a CSTD vial adapter and a CSTD syringe adapter. The syringe was separated from the vial, a CSTD needle adapter and 25g 5/8 "needle were added, and the syringe contents were discharged into a small beaker. The weight of the expelled drug product was converted to volume and subtracted from the 4.50mL vial fill volume. The hold-up volumes for 5 participants are presented in table 40. Of the 30 results produced, the average vial plus CSTD hold-up volume was 0.46mL with a standard deviation of 0.06mL. However, the highest hold-up volume for each of the 6 different CSTD ranges from 0.46mL to 0.58mL. The retention volume selected is 0.58mL +0.06mL = 0.64mL. At 3% filler variability, the target fill selected was (4.00 ml+0.64 mL)/0.97=4.78 mL.

Description and composition of commercial formulations

Commercial formulations for the 600 mg/vial (120 mg/mL) of nivolumab SC injection were sterile, non-pyrogenic, clear to rich milky white, colorless to yellow liquids. There may be a few particles whose appearance is consistent with proteinaceous particles. The pharmaceutical product is a disposable, preservative-free isotonic aqueous solution for Subcutaneous (SC) administration. Packaging Nawuzumab SC injection in 6R 1 flint glass vial with 20mm Daikyo D21-7SThe coated butyl plug was stoppered and secured with a 20mm aluminum seal with flip-off cap. The composition of the nivolumab SC injection is presented in table 41, which includes the quality criteria and function of each component. Overfill of the nivolumab SC injection was included in each vial to ensure that a 5.0mL amount of label could be administered to the patient.

^a Target filling bagIncluding 1.5mL overfill to compensate for vial, needle and syringe (VNS) hold-up, filling machine variability, and administration component hold-up. ^b Sodium chloride and histidine are present in rHuPH20 drug substance, but make insignificant contribution to the final composition. USP = united states pharmacopeia, ph.eur.= european pharmacopeia, NF = national formulary, q.s.= sufficient quantity

Selected physical and chemical characteristics

The selected physical and chemical properties of 150mg/mL of the nivolumab drug substance, 120mg/mL of the nivolumab drug product, and the dilution buffer are presented in Table 42.

^a The exact amounts of methionine and rHuPH20 in the dilution buffer will depend on the concentration of nivolumab used to make the drug substance for the following batch references: ELN A0C6F-067, ELN A0C6F-079, ELN A0C6F-083, ELN A0C6F-085-086.

Stress study

Short term room temperature and indoor illumination research

The goal of this study was to evaluate the effect of short-term room temperature/room illumination (RT/RL) exposure on the administration of nivolumab SC injections. The data generated in this short term study helps inform the length of exposure to be used in longer term stress studies. The drug product formulation used in this study was 120mg/mL of nivolumab in 20mM histidine buffer (pH 6.0) with 250mM sucrose, 0.05% w/v polysorbate 80, 50. Mu.M pentetic acid, 5mM methionine, and 2,000U/mL rHuPH 20. The bulk solution (5.67 mL aliquots) was filled into 10cc glass vials, which were stoppered and sealed. The vials were then subjected to the following stresses alone. Placing the vial in a horizontal position for worst case light exposure: 25C/RL exposure-test after 7 days, 14 days and 28 days at RT/RL; 25C/RL protected from light-7 days, 14 days and 28 days post-RT test (protected from light by wrapping the vials in aluminum foil).

The light source is a halophosphate bulb contained in a plastic tube. The unlabeled vials were placed on white paper in a horizontal position on a plastic tray. At each time point, the UV meter readings at all four corners were 0 μW/cm2. Photometer readings at the four corners were taken at each time point and ranged from 933 lux to 1023 lux. After withdrawal from exposure to stress conditions, the samples were stored at 5 ℃. All samples were tested together at the end of the study. Assays included appearance, pH, protein concentration, SE-HPLC, sub-visible particle levels (by HIAC), CE-SDS (R & NR), iCIEF, and enzyme activity.

Stability data from the study are presented in tables 43 to 45. As presented in table 43, the visual appearance of all samples at all time points was clear to pale milky, colorless to pale yellow solution. No changes in pH or protein concentration were observed in any of the samples in the 28 day study. The HMWS level of the sample stored at 25 ℃ accelerated light-shielding conditions increased by 0.12% and the HMWS level of the sample stored at 25 ℃ and 1,000 lux light increased by 1.00%. There was little change in LMWS levels from all samples to the 28 day time point.

^a Compliance = clear to pale milky, colorless to pale yellow solution.

Table 44 presents stability data for charge variants (by iCIEF) and enzyme activity. During the 28 day storage period, the acid species level of the sample stored in the dark at 25 ℃ increased by 1.0%, and the acid species level of the sample stored at 25 ℃/1,000 lux increased by 5.6%. The alkaline species level of both storage conditions was barely changed. The observed increase in acidic species was accompanied by a substantially equal decrease in the main peak area. The enzyme activity of the sample stored in the dark at 25℃was hardly changed, but the enzyme activity of the sample stored at 25℃at 1,000 lux was reduced by about 2% per day.

Table 45 presents data for CE-SDS reducing, CE-SDS non-reducing and sub-visible particulate matter. The percent purity as determined by reducing CE-SDS was unchanged over the 28 day study period, whether stored in light or dark. The percent purity, as determined by non-reducing CE-SDS, was reduced by 0.4% for both indoor illumination and light protected samples during the 28 day storage period. The level of sub-visible particles was low with no obvious trend.

Based on the results of this study, it is suggested to limit the exposure of the pharmaceutical product to indoor illumination.

Short term stability of rHuPH20 in Nawuzumab SC injection stored at 25-40 ℃

Studies were performed to evaluate the effect of storage temperature on rHuPH20 enzyme activity when the Nawuzumab SC injection was stored at a temperature ranging from 25℃to 40℃for up to 24 hours. Individual vials of the drug product were stored in the dark at 25 ℃, 32 ℃, 36 ℃ and 40 ℃ for up to 24 hours. After the shelf life, samples were tested for rHuPH20 activity. Each sample was tested in triplicate. Samples that have been stored continuously at 5 ℃ were also tested as controls. The results are presented in table 46.

Although the enzyme activity results showed significant variability, a trend in stability was observed to decrease with increasing temperature. Therefore, the storage time of the pharmaceutical product above 25 ℃ must be limited.

Physical stress study

Studies were performed to examine the effect of physical stress on stability of the nivolumab SC injection. The physical stresses imposed on a pharmaceutical product are similar to those that may be experienced during manufacture, transportation, or use in general. The stresses included freeze thawing (group 1), room temperature exposure, room illumination exposure, high temperature deflection (group 2), shock and vibration (group 3) and continuous storage at 5 ℃ protected from light (group 4-control). A batch of nivolumab SC injections was prepared and a 3.0mL aliquot of the drug product was filled into 6cc vials, which were then stoppered and sealed. The filled vials were subjected to various stresses and then placed in an accelerated steady state at 25 ℃.

For the freeze-thaw group of the study (group 1), filled vials were cycled between-20 ℃ and 5 ℃ four times with a minimum freeze time of 16 hours at-20 ℃ and a minimum thaw time of 8 hours at 5 ℃. It was observed that the solution in the vial was completely frozen after 2 to 3 hours of storage at-20 ℃ and completely thawed after 6 to 7 hours of storage at 5 ℃. For the room temperature, room illumination and high temperature offset group of the study (group 2), vials were stored at 25 ℃ for 22 days in the dark. Group 2 vials also underwent two shifts relative to this 25 ℃/dark storage condition: 72 hours at 25℃in 1,000 lux light (3 days), and 72 hours at 30℃in the absence of light (3 days). For shock and vibration stress (group 3), the vials were dropped five times from a height of 36 inches and then placed in a worst case horizontal position for 24 hours at 120rpm on an orbital shaker. The dropping and orbital shaking of the vials were performed at both 5 ℃ and ambient room temperature (about 22 ℃). Samples of drug product group 4 (i.e., control group) were kept at 5 ℃ for storage and protected from light.

Each set of samples was tested for solution appearance, pH, protein concentration, size homogeneity (by SE-HPLC) and sub-visible particulate matter (by HIAC) at the initial time points. In addition, charge variants were determined by iCIEF, molecular weight distribution was determined by CE-SDS (R & NR), and enzyme activity was determined using plate-based turbidimetry. The samples were retested after storage for 3 months and 6 months at 25 ℃ acceleration. Stability results are presented in tables 47-49.

^a Compliance = clear to pale milky, colorless to pale yellow solution.

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At the initial time point, no change in solution appearance, pH, or nivolumab concentration was observed in all four groups. Similar initial results for size homogeneity (by SE-HPLC), charge variants (by iCIEF), size variants (by CE-SDS (R & NR)) and enzyme activity were observed for groups 1, 3 and 4. The HMWS level of the group 2 samples (room temperature, room illumination and 30 ℃ storage) was 0.21% higher than the control, and the acidic species level of the group 2 samples was 1.4% higher than the control. The sub-visible particulate matter (via HIAC) was very low for all samples. At the initial time point, the enzyme activity of the group 2 samples was 2.5% lower than that of the control.

Timeout of cooling (Time Out) and Time under indoor lighting

The recommended storage condition for the Nawuzumab SC injection is 2-8deg.C, protected from light. Based on data collected from short-term room temperature/room lighting studies, short-term stability of rHuPH20 at 25-40 ℃ and physical stress studies, a timeout of refrigeration of the drug product and time under room lighting can be established. Short term RT/RL studies showed sensitivity to indoor lighting, rHuPH20 studies showed sensitivity of the enzyme to higher temperatures, and physical stress study data showed minimal effect of stress on quality attributes that may occur during pharmaceutical product manufacture, transportation or use. Based on the results of these studies, the time-out of refrigeration/time under indoor illumination (TOR/TARL) of the finished pharmaceutical product may be suggested. This TOR/TARL coverage begins with the application of the seal to the vial and eventually begins the period of preparation for administration to the patient, and includes post-filling activities (vial handling, inspection, sampling, labeling, secondary packaging, and shipping preparation) and temperature excursions during shipping until the beginning of preparation of a dose for patient administration.

The recommended storage condition for the Nawuzumab SC injection is 2-8deg.C, protected from light. Deviations from recommended storage conditions of up to 28 days at up to 25 ℃ are allowed, including up to 72 hours at room temperature and room illumination and up to 72 hours at 30 ℃.

Freezing temperature for pharmaceutical products in vials

As part of the physical stress evaluation, studies were conducted to determine the temperature at which the nivolumab SC injection frozen when filled in a 6cc glass vial and stored at subzero temperature for a relatively short period of time. Six vials of the nivolumab SC injection (3 mL/vial) were placed in a controlled temperature water bath. The bath temperature was lowered to-8 ℃ (temperature confirmed by calibrated thermocouple) and maintained for 9 hours. At the 9 hour time point, the vials were checked to determine if the solution in the vials had frozen. The bath temperature was then lowered to-10 ℃ and maintained for an additional 15 hours. After 15 hours at-10 ℃, the vials were again inspected to determine if the solution in the vials had frozen. The bath temperature was then lowered to-12 ℃ and maintained for 9 hours. After 9 hours of storage at-12 ℃, all 6 vials have been frozen.

The results of the study are presented in the following table. After 9 hours at-8 ℃ and 15 hours at-10 ℃ thereafter, all samples were still in solution. After 9 hours at-12 ℃, all six vials have been frozen. Thus, when filled in glass vials, the solution in the vials will not freeze when the freezing temperature of the nivolumab SC injection is between-12 ℃ and-10 ℃ and stored at temperatures as low as-10 ℃ for up to 15 hours.

Universal product information

Some general information about drug substances and drug products is provided below. The Nawuzumab drug substance was stored in 12L FFTp bags at-35℃and protected from light. The storage condition of the medicine product is 2-8deg.C, and the medicine product is protected from light. The compositions of the drug substance and drug product are listed in table 51 and the selected properties of the drug substance, drug product and dilution buffer are presented in table 52.

^a Methionine and rHuPH20 were added to the dilution buffer, which was then added to the drug substance to produce the drug product. ^b Sodium chloride and histidine are present in rHuPH20 drug substance, but make insignificant contribution to the final composition

Example 2-subcutaneous Nawuzumab with or without rHuPH20

In ongoing phase 1/2 studies, the PK, safety, efficacy and tolerability of nivolumab monotherapy with or without Subcutaneous (SC) administration of hyaluronidase rHuPH20 in patients was evaluated in solid tumors (metastatic melanoma, RCC, NSCLC, HCC and CRC) that had well characterized PK, efficacy, safety and immunogenicity of nivolumab after IV administration. Other solid tumors (esophageal gastric junction [ GEJ ], gastric Cancer (GC), metastatic urothelial cancer (mUC), and SCCHN that adequately characterize PK of IV nivolumab are permitted).

The initial SC dose selected for part A was 720mg Q4W. Based on the preliminary PK from part a and the subsequent modeling, this study was continued as planned with a second dose of 960mg q4w for part B.

For part a and part B, PK of single dose SC nivolumab (with and without rHuPH 20) was characterized, followed by characterization of 480mg q4w of IV nivolumab at week 4. These SC PK data are used to upgrade existing IV PPK models. A dosing regimen of 1200mg q4w of SC nivolumab was then selected using the combined SC/IV PPK model for use in subsequent studies.

Part C and part D will provide additional PK and safety data after administration of 1200mg q4w in part C (about 45 patients) and part D (about 36 patients). Part C was designed to characterize PK and study safety for continuous administration of 1200mg q4w of SC nivolumab with transition from IV (transition from part a and part B). Part D includes PK and safety of continuous administration of SC nivolumab 1200mg q4w from the start of therapy. Intermediate analysis will be performed, including evaluation of pre-dose cycle 2 day 1 PK and early safety in approximately 10 subjects using a 1200mg dose.

The main objective of part a-part D was to describe PK of SC nivolumab with or without rHuPH20 as assessed by various metrics including Cavgd28, cmind28 and Cmax 1.

The main objective of the part a-part D study was to describe the pharmacokinetics of subcutaneously administered nivolumab with or without rHuPH20, and the endpoints were Cmax, tmax, AUC (TAU), and Ctau (parts A, B and D) and Ctau (part C). Secondary objectives include (i) assessing the safety profile of SC nivolumab; (ii) Evaluating the incidence of AEs in widely Standardized MedDRA Queries (SMQ) of allergic reactions and selected AE hypersensitivity/infusion reaction categories; and (iii) assessing the immunogenicity of the nivolumab. Secondary endpoints include (i) incidence of AE, SAE, AE leading to discontinuation, death, and laboratory abnormalities; (ii) Incidence of AEs in broad SMQ of allergic reactions occurring within 2 days after study drug administration; (iii) The hypersensitivity/infusion reactions occurring within 2 days after study drug administration selected the incidence of events within the AE category; and (iv) the incidence of anti-nivolumab and neutralizing antibodies, if applicable. Exploratory targets include (i) evaluating preliminary efficacy in all participants; (ii) Biomarker measures characterizing immune function and tumor genetics and genomics; (iii) assessing the immunogenicity of rHuPH 20; and (IV) assessing the primary participant experience and preference for SC or IV administration of nivolumab. Exploratory endpoints include (i) ORR, PFS, and OS; (ii) A generalized measure of the change (or% change) in various biomarkers and molecular characteristics of a tumor from baseline; (iii) Incidence of anti-rHuPH 20 antibodies and neutralizing antibodies, if applicable; and (iv) patient experience/preference questionnaires and qualitative patient interviews.

Results

Thirty-two subjects have been treated with a single dose of nivolumab (720 mg or 960 mg) administered via SC injection co-administered with rHuPH20 followed by IV nivolumab. Intermediate analysis was performed after all subjects in part B (group 3) completed 1 cycle of SC nivolumab. PK and early safety data were analyzed for 22 subjects in part a-group 1 (720 mg+rhuph 20) and 10 subjects in part B-group 3 (960 mg+rhuph 20). There is no efficacy form output available when locked due to short-term follow-up.

Median follow-up to DBL (minimum pre-dose cycle 2 day 1 in all part B groups 3), part a-group 1 (n=22) was 4.4 months (range: 2.4-8.8 months), and part B-group 3 (n=10) was 2.5 months (range: 2.1-3.5 months).

Baseline disease characteristics of study participants are shown in the table

53. There were 14 men and 18 women. The median (range) age for all patients was 66.5 (48-90) years. The 32 subjects represented different patient populations, including subjects with a range of ages, weights and tumors (NSCLC, CRC, RCC, HCC, melanoma and SCCHN) in an advanced/metastatic setting.

Table 53A: baseline characteristics

Preliminary security data

Group 1 (720 mg+rhuph20 SC dose and IV dose thereafter): any grade of treatment-related AE (TRAE) was reported in 10 (45.5%) subjects and was an AE commonly known within the nivolumab IV program (table 54B). Low grade erythema, irritation and swelling at the SC injection site were reported in 3 (14%) subjects.

Group 3 (960 mg+rhuph20 SC dose and IV dose thereafter): any grade of TRAE was reported in 3 (30%) subjects, including grade 1-2 immune-mediated rashes, reticulocytes, and local site reactions. Low grade erythema, itching and swelling at the SC injection site were reported in 2 (20%) subjects. At DBL, no treatment-related SAE (TRSAE) was reported in group 3.

Early safety analysis of subjects receiving SC nivolumab 720mg+rhuph20 (group 1) and 960mg sc+rhuph20 (group 3) was descriptive (and not intended for inter-group comparison). The clinical safety profile of single dose SC nivolumab and IV following (circulating 2+) reflects treatment-related AEs and SAE previously reported within nivolumab IV IB, with the exception of SC local response. With regard to local AEs using SC injection, there was no unexpected local site reaction, and all reported events were low-grade and manageable. All 32 subjects in groups 1 and 3 received a circulating 1 day 1 SC nivolumab (720 mg or 960 mg) +rhuph20 dose of 6mL or 8mL, respectively, administered in a single manual injection.

Two deaths occurred during the study due to disease progression (neither attributed to study drug).

AE. The summaries of TRAE and TRSAE are provided in tables 2A-2C, respectively.

Table 54A: adverse event summary-all treated subjects, group 1 and group 3-intermediate analysis

^a 5 stages: men aged 50 die from malignant tumor progression, RCC with lung metastasis; and death of 76 year old men due to malignancy progression, CRC

Table 54B: summary of drug-related adverse events on worst CTC scale-all treated subjects, group 1 and group 3-intermediate analysis

Table 54C: summary of drug-related severe adverse events-all treated subjects, group 1 and group 3-intermediate analysis

Population pharmacokinetic analysis of combined nivolumab SC/IV data

Population Pharmacokinetic (PPK) modeling and simulation methods were employed to characterize SC nivolumab PK and optimize dose selection for SC nivolumab. The goal of this modeling-based analysis was to construct a PPK model to describe the nivolumab concentration data when administered by both SC and IV routes of administration. The nivolumab concentration data following the first dose SC administration from run trial portions a and B can be obtained from 29 subjects in 2 dose levels including 720mg SC nivolumab + rHuPH20 (portion a-group 1) and 960mg SC nivolumab + rHuPH20 (portion B-group 3). These SC data were combined with existing IV concentration data to develop a combined SC/IV PPK model for nivolumab. The extravascular absorption component is added to the existing established IV PPK model, followed by estimation of appropriate parameters of absorption, including BA (F1) and absorption rate constant (ka). Estimates of PK parameters from the combined SC/IV model are summarized in table 55.

Table 55: estimates of PK parameters from a combined SC/IV PPK model of nivolumab

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^a Parameters with fixed values (non-estimated) are denoted by the superscript "f" after the name, the fixed values of which are given in the estimated value column. ^b The random effect and residual parameters containing the colon (:) represent the relevant parameters. ^c The random effect and residual parameter estimates are shown as the variance (standard deviation) of the diagonal elements (ωi, i or σi, i) and the covariance (correlation) of the off-diagonal elements (ωi, i or σi, i). ^d RSE% is the relative standard error (standard error as a percentage of the estimated value). ^e The random effect and confidence interval of the residual parameter are for variance or covariance. ^BS Confidence interval values were calculated by self-help (934 out of 1000 total success).

The structural PK model consists of 2 compartments: zero order absorption for IV administration and first order absorption for SC administration. Model determination of Nawuzumab was 67% BA with high accuracy (95% CI:60% -75%). PPK modeling and simulation methods were used to characterize SC nivolumab PK and optimize dose selection for SC nivolumab. The goal of this modeling-based analysis was to construct a PPK model to describe the nivolumab concentration data when administered by both SC and IV routes of administration. The nivolumab concentration data following the first dose SC administration from the ongoing trial (part a and part B) can be obtained from 29 subjects in 2 dose levels including 720mg SC nivolumab + rHuPH20 (part a-group 1) and 960mg SC nivolumab + rHuPH20 (part B-group 3). These SC data were combined with existing IV concentration data to develop a combined SC/IV PPK model for nivolumab. The extravascular absorption component is added to the existing established IV PPK model, followed by estimation of appropriate parameters of absorption, including BA (F1) and absorption rate constant (ka). Estimates of PK parameters from the combined SC/IV model are summarized in table 55.

The structural PK model consists of 2 compartments: zero order absorption for IV administration and first order absorption for SC administration. Model determination of Nawuzumab was 67% BA with high accuracy (95% CI:60% -75%). PPK modeling and simulation methods were used to characterize SC nivolumab PK and optimize dose selection for SC nivolumab. The goal of this modeling-based analysis was to construct a PPK model to describe the nivolumab concentration data when administered by both SC and IV routes of administration. The nivolumab concentration data following the first dose SC administration from ongoing CA2098KX (part a and part B) was obtained from 29 subjects in 2 dose levels including 720mg SC nivolumab + rHuPH20 (part a-group 1) and 960mg SC nivolumab + rHuPH20 (part B-group 3). These SC data were combined with existing IV concentration data to develop a combined SC/IV PPK model for nivolumab. The extravascular absorption component is added to the existing established IV PPK model, followed by estimation of appropriate parameters of absorption, including BA (F1) and absorption rate constant (ka). Estimates of PK parameters from the combined SC/IV model are summarized in table 55.

The structural PK model consists of 2 compartments: zero order absorption for IV administration and first order absorption for SC administration. Model determination of Nawuzumab was 67% BA with high accuracy (95% CI:60% -75%).

The combined SC/IV PPK model underwent internal validation practices to ensure that the model was able to predict concentration values observed after SC administration. Visual predictive examination (fig. 4) showed that the combined SC/IV model adequately captured and described the observed SC nivolumab concentration data.

Using the above model, deterministic simulations were performed to generate exposure metrics for subjects in CA2098KX that were treated with PK data. The exposure metrics are provided in table 56 as a summary of dose levels.

Table 56: summary of predicted exposures by dose level in treated subjects

Abbreviations: cavgd28=average concentration at day 28; cmax1 = maximum concentration after the first dose; cmind28 = minimum concentration at day 28; CV = coefficient of variation; geoMean = geometric mean; q4w=every 4 weeks; sc=subcutaneous; SD = standard deviation; tmax1=time after the first dose to maximum plasma concentration.

Single group expanded cohort of RCC subjects

The trial will be expanded to include a single group, single tumor expansion group (part E) with advanced/metastatic RCC (fig. 3). The main objective of part E was to demonstrate the PK non-inferior efficacy of SC nivolumab 1200mg+rHuPH20 (coformulation) Q4W compared to IV administration (3 mg/kg Q2W) by comparing model predicted SC and IV exposures. Non-inferior efficacy is defined as a geometric mean ratio of exposure measures with a bilateral 90% CI of at least 0.8 (common primary endpoint: cavgd28; cmnd 28). Demonstration of non-inferior efficacy of Cavgd28 and Cmind28 will ensure that the efficacy of SC nivolumab Q4W will be maintained at a level comparable to IV nivolumab 3mg/kg Q2W.

The secondary objective of part E was to (i) evaluate PK of SC nivolumab co-formulated with rHuPH 20; (ii) Evaluating the safety profile of SC nivolumab co-formulated with rHuPH 20; (iii) evaluating the immunogenicity of the nivolumab; and (iv) evaluating the response assessed by the researcher. Secondary endpoints include (i) Cmax1, AUC (TAU), cavg (ss), and Cmin (ss); (ii) Incidence of AE, SAE, AE that caused discontinuation, death, and laboratory abnormalities; (iii) The incidence of anti-nivolumab antibodies and neutralizing antibodies, if applicable; and (iv) ORR. Exploratory goals include (i) exploring efficacy in all participants; (ii) Explore biomarker measures of immune function and tumor genetics and genomics; (iii) exploring the immunogenicity of rHuPH 20; and (iv) explore participant experiences using SC nivolumab. Exploratory endpoints include (i) PFS, OS, time to reaction, and duration of reaction; (ii) Changes in different biomarkers and molecular characteristics of tumor/blood from baseline; (iii) Incidence of anti-rHuPH 20 antibodies and neutralizing antibodies, if applicable; and (iv) patient experience/preference questionnaires and qualitative patient interviews.

The selection of RCC as a tumor type for part E is supported by the extensive historical PK data of IV nivolumab of tumor type and the acknowledged PPK and E-R models. The E-R curve for efficacy and safety of nivolumab in RCC was flat over the dose range of 1mg/kg to 10mg/kg (data not shown). Given the well-established flat E-R relationship of RCC and melanoma over a broad range of test doses, the benefit/risk assessment for SC nivolumab in part E can reasonably support extrapolation to all tumor types for which IV nivolumab has been demonstrated to be safe and effective. The selection of 3mg/kg IV Q2W as the actual IV treatment group is based on: robust data on exposure, efficacy and safety of 3mg/kg IV Q2W. Most studies (including critical studies for all approved indications) evaluate nivolumab at a dose of 3mg/kg IV Q2W. Thus, well-characterized efficacy, safety and PK profile of the dose can be obtained. The acknowledged E-R across the 3mg/kg IV Q2W regimen in RCC and melanoma showed that exposure was not significantly associated with safety/efficacy. The demonstration of PK non-inferior efficacy of SC dose compared to 3mg/kg Q2W IV dose is sufficient to conclude that the benefit-risk profile of SC nivolumab will be comparable to IV nivolumab in advanced/metastatic RCC and by extrapolation to other tumor types for the following reasons:

Efficacy of 3mg/kg IV Q2W has been demonstrated in a dose range and in critical studies across tumor types. Thus, if similar or larger Cavgd28 and Cmind28 were achieved with an SC dosing regimen of 1200mg q4w, it is expected that the safety and efficacy profile of SC nivolumab would be comparable to IV nivolumab.

Inclusion/exclusion criteria/patient characteristics

The key inclusion criteria include: (i) untreated with PD-L1; (ii) Histological confirmation of RCC with clear cell components (must have received at least 1 and no more than 2 previous systemic treatment protocol lines in an advanced or metastatic setting, and must have evidence of progression within 6 months at or after the last treatment protocol was received and before study into the group); (iii) Formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks or unstained slides (archival or near-term) of tumor samples that require the subject for biomarker evaluation upon entry into the study; (iv) Male and female participants must be 12 years old or age at informed consent; (v) The participants must be assessed for tumor PD-L1 expression by Immunohistochemistry (IHC); (vi) Diseases measurable according to the standard of solid tumor Response Evaluation (RECIST) version 1.1; (vii) participants must have ECOG physical status of 0 or 1; and (viii) all participants must have the ability to follow-up therapy, patient report outcome, PK, pharmacodynamic sample collection, and study follow-up requirements.

Critical exclusion criteria included (i) treatment with a botanical formulation (e.g., herbal supplement or traditional chinese medicine) within 2 weeks prior to randomization/treatment to treat the disease under study; (ii) Participants had active autoimmune disease or any other condition requiring systemic treatment with corticosteroid (> 10mg daily prednisone equivalent) within 14 days of randomization or systemic treatment with other immunosuppressive drugs within 30 days of randomization (in the absence of active autoimmune disease, inhalation or topical steroids were allowed, and an adrenal replacement steroid dose of >10mg daily prednisone equivalent); (iii) Participants with type I diabetes, hypothyroidism requiring only hormone replacement, skin disorders that do not require systemic treatment (such as vitiligo, psoriasis or alopecia), or conditions that are expected to not relapse in the absence of external triggers are allowed to enter the group; (iv) Untreated symptomatic Central Nervous System (CNS) metastasis (if CNS metastasis is asymptomatic and does not require immediate treatment, or has been treated and the patient has been neurological restored to baseline status (except for residual signs or symptoms associated with CNS treatment), then the patient is eligible, otherwise, the patient must have been off corticosteroid prior to group entry, or is taking a steady or reduced dose of +.10 mg daily prednisone (or equivalent) for at least 2 weeks); (v) Patients carrying known Human Immunodeficiency Virus (HIV) who have had Acquired Immune Deficiency Syndrome (AIDS) defined opportunistic infections in the past year, or current group 4 (CD 4) count <350 cells/uL; (vi) organ dysfunction based on baseline laboratory assessment; (vii) Patients with concurrent malignancy in need of treatment (patients with previously treated malignancy are eligible if treatment has been completed at least 2 years prior to registration and the patient has no evidence of disease; and patients with concurrent malignancy that are clinically stable and do not require treatment for the tumor are also eligible); (viii) Patients with severe or uncontrolled medical disorders; (ix) Serological evidence of chronic Hepatitis B Virus (HBV) infection, and HBV viral load is above the limit of quantification. Participants with chronic HBV infection are necessarily undergoing concurrent viral suppression therapy; (x) Current serological evidence of Hepatitis C Virus (HCV) infection, with HCV viral load above the limit of quantification; (xi) Participants who had received live/attenuated vaccine within 30 days of the first treatment; (xii) Has allergic reaction or hypersensitivity history to the research drug component; and (xiii) previous treatments with anti-PD-1, anti-PD-L1, anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies or any other antibody or drug that specifically targets a T cell costimulatory or checkpoint pathway.

Principle of selection of SC dose

Dose selection for SC nivolumab was performed using PPK modeling and simulation methods. The nivolumab concentration data following administration of the first dose SC from part a and part B were collected in2 dose levels (720 mg and 960 mg) co-administered with rHuPH 20. These were combined with existing IV concentration data to develop a combined SC and IV PPK model for nivolumab. The extravascular absorption component is added to the existing established IV PPK model, followed by estimation of appropriate parameters of absorption, including BA and ka. Simulations were performed using this combined SC/IV PPK model to predict systemic exposure after a series of doses administered over a range of body weights. It was determined under various scenarios that explain the potential uncertainty of the tested BAs that the SC nivolumab 1200mg Q4W regimen would elicit a benefit-risk profile comparable to IV nivolumab 3mg/kg Q2W. This is based on the following principle: for all body weights, this dosing regimen was able to deliver Cavgd28 and Cmin28 exposures similar to or greater than Cavgd28 and Cmin28 exposures associated with IV nivolumab 3mg/kg Q2W, while maintaining all exposure metrics below those associated with IV nivolumab 10mg/kg Q2W. In addition, clinical trial simulations under the most conservative scenario considered (low bioavailability, in RCC populations that tend to have higher body weight) indicate that in SC versus IV trials, SC nivolumab 1200mg q4w has a relatively higher probability than 960mg q4w to meet non-poor efficacy criteria.

Regarding the expected safety profile with a 1200mg SC dose, it is expected that the predicted exposure will not exceed those produced by 10mg/kg Q2W IV of nivolumab (the safety of this dose has been well characterized and shows a similarity to 3mg/kg Q2W IV). Whereas there is no clinically significant difference in AE incidence between IV exposures of 3mg/kg and 10mg/kg Q2W IV, 1200mg+rHuPH20 Q4W SC is a suitable dose that will provide an exposure equal to or greater than 3mg/kg Q2W IV and within the exposure produced by 10mg/kg Q2W IV. This will be evaluated as part of the ongoing correction to characterize the actual PK of the 1200mg SC dose (part C and part D) and the preliminary results will be reviewed before starting part E.

A further comparison of the deterministic simulated exposures from patients in groups a and B was performed to determine if there was any difference in exposure of 1200mg Q4W of SC nivolumab relative to 3mg/kg Q2W based on tumor type. These simulations explain the absorption, bioavailability, systemic PK parameters, and individual parameters of individual covariate information. There was no apparent trend or difference in the Geometric Mean Ratio (GMR) for all exposure metrics among the tumor types collected in this study. All GMRs were equal to or greater than 1, indicating that the tumor type was unlikely to affect the interpretation of non-bad exposure, and body weight would continue to remain as a major contributor to any difference in exposure from SC 1200Q4W and 3mg/kg Q2W IV dosing (fig. 5A-5C).

Example 3-subcutaneous Nawuzumab with or without rHuPH20

With the support of clinical studies using Subcutaneous (SC) administration of nivolumab, non-clinical studies were performed in cynomolgus monkeys to evaluate local tolerance to nivolumab and systemic exposure when administered twice (3 weeks apart) as SC formulations with and without rHuPH 20. SC nivolumab was supplied at 154.57mg/mL and was administered twice (day 1 and day 22/day 20 [ male/female ]) by SC injection to groups of 3 monkeys per individual at doses of 0mg/kg (vehicle), 50mg/kg (without rHuPH 20) or 50mg/kg (with rHuPH20, 2000U/mL). All doses were administered at 0.5mL/kg in a vehicle/carrier (histidine buffer) consisting of 20mM histidine, 250mM sucrose, 0.05% polysorbate 80 and 50 μm pentetic acid. Samples for toxicological analysis were collected after dosing on day 1 and scheduled necropsies were performed 72 hours after dosing on day 22/day 21 (male/female).

The average total body exposure of nivolumab (AUC [0-T ]) at 50mg/kg with rHuPH20 was generally similar to that without rHuPH20 (Table 57). The mean to maximum plasma concentration time (Tmax) with or without rHuPH20 was 32 hours or 68 hours after dose, respectively. These results demonstrate that while Tmax with rHuPH20 co-administration was earlier in this non-clinical study, rHuPH20 had no significant effect on overall nivolumab systemic exposure.

Table 57: summary of toxicological kinetics-average sex combination value

Abbreviations: area under the curve of AUC = concentration-time curve; cmax = maximum concentration; tmax = time to maximum plasma concentration. ^a Values were calculated with all monkeys/exclusions of monkeys with detectable anti-nivolumab antibodies present in treatment.

The appearance of the nivolumab anti-drug antibodies (ADA) in treatment was detected in 1 out of 6 monkeys with or without rHuPH 20. However, in general, the presence of ADA present in the treatment has no significant effect on the exposure of nivolumab.

The administration of nivolumab at 50mg/kg with and without rHuPH20 (2000U/mL) was well tolerated, no clinical observations and no local tolerability problems at the SC injection site, and in this non-clinical study there was no meaningful difference in the exposure to nivolumab with or without rHuPH 20.

Example 3-subcutaneous Nawuzumab in combination with recombinant human hyaluronidase in advanced, recurrent or metastatic non-small cell lung cancer with previous treatment

Phase 3 studies will be performed to evaluate the administration of Subcutaneous (SC) nivolumab co-formulated with recombinant human PH20 (rHuPH 20) compared to Intravenous (IV) nivolumab in participants with advanced, recurrent or metastatic non-small cell lung cancer (NSCLC) previously treated. This study was sought to establish the Pharmacokinetic (PK) of 1200mg of nivolumab co-formulated with 20,000 units rHuPH20 administered every 4 weeks (Q4W) SC and the efficacy non-inferior compared to 3mg/kg of nivolumab administered every 2 weeks (Q2W) IV. Throughout this protocol, the co-formulation of nivolumab and rHuPH20 will be referred to as SC nivolumab, and the IV formulation of nivolumab will be referred to as IV nivolumab.

Men or women 18 years old or older (or most of the local age) have histologically confirmed stage IIIB/IIIC/IV NSCLC (squamous or non-squamous) that has undergone disease recurrence or progression during or after 1 prior systemic therapy for advanced or metastatic disease. Targets and endpoints are presented in table 58.

Table 58: target and endpoint

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Abbreviations: AE = adverse event; BICR = blind independent central screening; cmind28 = minimum serum concentration over 28 days; cavgd28 = mean serum concentration at day 28; cmax1 = maximum serum concentration after the first dose; cmins = steady state trough concentration; cavgss = steady state mean serum concentration; DCR = disease control rate; doR = duration of reaction; IV = intravenous; medDRA = international medical dictionary of terms (Medical Dictionary for Regulatory Activities); ORR = objective response rate; OS = total survival; PFS = progression free survival; PK = pharmacokinetics; q2w=every 2 weeks; q4w=every 4 weeks; rhuph20=recombinant human hyaluronidase PH20; SAE = severe adverse event; sc=subcutaneous; SMQ = standardized med dra query; tmax = time to Cmax 1; TTR = time to reaction.

Overall design

The present study is a multicentric, randomized, open-label, phase 3 study that will evaluate the PK and efficacy non-inferior efficacy of SC nivolumab compared to IV nivolumab, as well as the safety and tolerability of SC nivolumab in participants with advanced, recurrent, or metastatic NSCLC.

Group a (n=257): 1200mg of a co-formulation of nivolumab with 20,000 units of SC nivolumab of rHuPH20, Q4 W.+ -.7 days; and

group B (n=257): IV nivolumab 3mg/kg, Q2 W.+ -. 3 days (FIG. 6).

A total of about 514 participants will be randomized in a 1:1 fashion into the following treatment groups: randomization treatment groups will be stratified with histological (squamous versus non-squamous), PD-L1 scores (> 1% and <1% and indeterminate), body weight (< 65kg and 65-90kg and >90 kg), and eastern tumor co-operating group physical stamina ECOG-PS (0 and 1) (fig. 6). ECOG PS must be assessed within 14 days prior to randomization. Dosing in this study will continue until the study is terminated by the first occurrence, based on the solid tumor Response Evaluation Criteria (RECIST) v.1.1 disease progression, unacceptable toxicity, withdrawal consent, 104 weeks of treatment completion, death, or sponsor.

The tumor assessment in the study should consist of CT/MRI of contrast-enhanced CT of the chest, abdomen, pelvis and all other known and/or suspected diseased sites, should be performed every 8 weeks (+ -7 days) from randomization for 2 years (104 weeks), then every 12 weeks (+ -7 days) until disease progression and treatment discontinuation (including post-progression treatment) (later, the latter occurrence is the subject). Partial Response (PR) and Complete Response (CR) must be assessed and confirmation must be made at least 4 weeks after initial assessment. Tumor response will be assessed using RECIST 1.1.

Serial blood samples will be collected during cycle 1 in group a and during cycles 1 and 2 in group B, followed by pre-dose PK samples during the entire treatment period in both group a and group B to characterize PK and immunogenicity of nivolumab.

Safety monitoring will consist of physical examination, vital sign measurement and clinical laboratory assessment at selected times throughout the dosing interval. The AE of the participants will be closely monitored throughout the study. Collecting AEs and severity according to the national cancer institute common adverse event terminology standard (NCI CTCAE) v.5 standard will also include local injection site reactions and IV infusion-related reactions following SC administration.

Dose validity

The absorption profile of nivolumab at subcutaneous administration was characterized using PK data from participants of the study (in which nivolumab (720 mg, 960mg, and 1200 mg) was SC administered with or without rHuPH 20) and historical IV data for several tumor types (data from approximately 3000 patients). Population PK (PPK) analysis estimated an average (90% CI) bioavailability of 70% (66-74%) and a first order absorption rate of 0.250 (90% CI: 0.225-0.274) day-1. The effect of all other PK parameters and covariates on these parameters were consistent with those previously estimated using the IV PPK model. Model estimated exposures using SC nivolumab 1200mg Q4W and IV nivolumab 3mg/kg Q2W are presented in table 59. Based on these results, a dose of 1200mg of Q4W nivolumab SC nivolumab was expected to provide similar or higher exposure over all body weight ranges as compared to 3mg/kg Q2W of IV nivolumab. Geometric mean exposure using 1200mg Q4W SC administration of nivolumab was also lower than that of the highest tolerated IV dose of 10mg/kg Q2W and was considered safe (fig. 7).

Table 59: model estimated exposure to SC (with rHUPH 20) and IV nivolumab

Abbreviations: cavgd28 = mean serum concentration at day 28; cmax1 = maximum serum concentration after the first dose; cmind28 = minimum serum concentration at day 28; CV = coefficient of variation; IV = intravenous; q4w=every 4 weeks; q2w=every 2 weeks; sc=subcutaneous.

Criteria for inclusion

Participants must have histologically confirmed NSCLC (squamous or non-squamous) and exhibit stage IIIB/IIIC/IV disease (according to international lung cancer research association chest tumor staging manual 8 th edition) or exhibit relapse or disease progression (according to RECIST 1.1 criteria) following multimode therapy (radiation therapy, surgical excision or deterministic radiotherapy for locally advanced disease). The participants had to have a disease measurable by CT or MRI according to RECIST 1.1 criteria within 28 days prior to the first therapeutic dose. Participants had to have ECOG physical status of 0 or 1 assessed within 14 days of randomization. The participants must have experienced disease recurrence or progression during or after 1 prior systemic therapy for advanced or metastatic disease.

Maintenance therapy following platinum-based dual drug chemotherapy is not considered a separate treatment regimen. Participants who received pemetrexed, bevacizumab, or erlotinib as maintenance therapy (non-progressors of platinum-based dual drug chemotherapy) and developed progress were eligible. Participants who had received adjuvant or neoadjuvant platinum dual drug chemotherapy (after surgery and/or radiation therapy) and had developed recurrent or metastatic disease within 6 months of completion of the therapy were eligible. Participants who had recurrent disease >6 months after adjuvant or neoadjuvant platinum-based chemotherapy, and who subsequently also developed during or after the platinum double dosing regimen administered for treatment of recurrence, were eligible.

All participants with non-squamous histology must be tested for EGFR mutation status (including but not limited to deletions in exon 19 and exon 21[ L858R ] substitutions); the use of institutional approved testing is greatly encouraged. Participants positive for sensitized EGFR mutations should develop disease progression after receiving a previously approved EGFR inhibitor. Participants with non-squamous histology with known ALK translocations should develop disease progression after receiving a previously approved ALK inhibitor. The ALK mutation test is not required for this study.

Participants with symptomatic tumor lesions at baseline who may need palliative radiation therapy within 4 weeks of the first dose of study treatment are greatly encouraged to receive palliative radiation therapy prior to group entry. Palliative radiation therapy should be completed 2 weeks before the first dose. If there is a recorded (radiographic) disease progression in the previously irradiated region (according to RECIST 1.1 criteria), then the target lesion may be located at that site.

Participants with Chronic Obstructive Pulmonary Disease (COPD) controlled at the time of study entry were eligible.

Formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks or a minimum of 20 unstained tumor tissue slides obtained from core biopsies, drill biopsies (punch biopsys), resected biopsies, or surgical samples prior to group entry (within 12 weeks of group entry) were not subjected to intermediate systemic anti-cancer treatment between the time of acquisition in IRT and treatment randomization. If a minimum of 20 slides are not available regardless of effort, fewer slides may be acceptable in some cases after discussion with a medical supervisor or a designated person. If tumor tissue obtained within 12 weeks of the entry group is not available, then archival tissue pieces can be submitted (within about 12 months of the entry group) without intermediate systemic anti-cancer treatment between the time of acquisition in IRT and treatment randomization. Fine needle aspirates and other cytologic samples are not acceptable. Assessment of tumor cell PD-L1 expression by IHC must be performed by an analytical laboratory using pre-treatment tissue samples. The analytical laboratory must provide IRTs with PD-L1 results prior to randomization.

Exclusion criteria

Participants had active autoimmune disease or any other condition requiring systemic treatment with corticosteroid (> 10mg daily prednisone equivalent) within 14 days of randomization or systemic treatment with other immunosuppressive drugs within 30 days of randomization. In the absence of active autoimmune disease, inhaled or topical steroids are allowed, as well as adrenal replacement steroid doses of >10mg daily prednisone equivalent. A known history of positive tests for Human Immunodeficiency Virus (HIV) or a known Acquired Immune Deficiency Syndrome (AIDS).

Participants with type I diabetes, hypothyroidism requiring only hormone replacement, skin disorders that do not require systemic treatment (such as vitiligo, psoriasis or alopecia), or conditions that are expected to not relapse in the absence of external triggers are allowed to enter the group.

Participants with untreated symptomatic CNS metastases. Participants are eligible if CNS metastasis is asymptomatic and does not require immediate treatment, or has been treated and the patient is neurological restored to baseline status (except for residual signs or symptoms associated with CNS treatment). In addition, the participants must have been out of the corticosteroid prior to entering the group, or are taking a steady or reduced dose of +.10 mg daily prednisone (or equivalent) for at least 2 weeks.

Participants with a concurrent malignancy in need of treatment. Participants with previously treated malignancy were eligible if treatment was completed at least 2 years prior to randomization and the patient had no evidence of disease. Patients with concurrent malignancies that are clinically stable and do not require treatment for tumors are also eligible.

Participants with interstitial lung disease that are symptomatic or may interfere with the detection or management of suspected drug-related pulmonary toxicity.

Participants in the study who had received treatment with botanical preparations (e.g., herbal supplements or traditional chinese medicines) within 2 weeks prior to randomization/treatment to treat the disease. Participants who had received live/attenuated vaccine within 30 days of the first treatment. Participants who have received prior treatment with anti-PD-1, anti-PD-L1, anti-CTLA 4 antibodies, or any other antibody or drug that specifically targets the T cell costimulatory or checkpoint pathways.

Organ dysfunction based on baseline laboratory assessment included (i) White Blood Cells (WBCs) <2000/μl; (ii) neutrophils <1500/μl; (iii) platelets <100x 103/. Mu.L; (iv) hemoglobin <9.0g/dL; (v) Serum creatinine >1.5x upper limit of healthy population (ULN) unless creatinine clearance is greater than or equal to 40mL/min (measured or calculated using the Cockroft-Gault formula); (vi) Glutamic-oxaloacetic transaminase (AST)/glutamic-pyruvic transaminase (ALT): 3.0 XULN; (vii) Total bilirubin >1.5x ULN (except for participants with gilbert syndrome, the total bilirubin level must be <3.0x ULN); and (viii) any positive test result of HBV or HCV virus that indicates the presence of virus, e.g., positive for hepatitis b surface antigen (HBsAg, australian antigen), or positive for hepatitis c antibody (anti-HCV) (except HCV-RNA negative).

Subcutaneous administration of nivolumab with rHuPH20 (group a)

SC nivolumab should be administered every 4 weeks ± 7 days on day 1 of each treatment cycle until progression, unacceptable toxicity, withdrawal of consent, treatment completion, death or end of study for 104 weeks (2 years), whichever occurs first. Participants should begin study treatment within 3 calendar days of randomization.

The dose of nivolumab will not be allowed to increase or decrease stepwise. Participants may be dosed no less than 25 days from the previous dose of the Q4W cycle. For the first dose of nivolumab, no precursor drug was suggested.

The dose of nivolumab may be discontinued, delayed or discontinued depending on the tolerance of the participant to the treatment. The dosing visit was not skipped, only delayed.

For the first cycle, SC nivolumab will be administered via manual injection in one quadrant of the abdomen. For the subsequent cycle, one of the four quadrants of the abdomen and either thigh are options and the injection sites should alternate. SC administration should be as smooth as possible (i.e., not start or stop, and at a steady rate) over a period of about 3-5 minutes. Participants will be monitored for approximately 360 minutes after manual SC-nivolumab injection on cycle 1 day 1 and cycle 2 day 1. Participants receiving subsequent SC injections may be monitored for about 30 minutes following the injection, as desired clinically and as deemed appropriate by the researcher. In addition, all participants will be contacted approximately 24 hours after each SC injection for reporting any injection site reactions. The injection site will also be evaluated at the next study visit. The SC injection site, duration and needle type must be recorded on an electronic case report table (eCRF). Instructions for preparing SC nivolumab doses are provided in the Pharmacy Manual (Pharmacy Manual).

Example 4-subcutaneous Nawuzumab with or without rHuPH20

In ongoing phase 1/2 studies, patients (pt) with unused checkpoint inhibitors of metastatic/unresectable solid tumors and measurable disease were administered different doses of nivolumab (with or without rHuPH 20) to ≡18 years of age, ECOG PS 0-1. The main objective was to describe subcutaneous nivolumab Pharmacokinetics (PK); and secondary objectives are safety and immunogenicity. Additional analysis compares exposure to historical IV nivolumab. In cycle 1, patients in part a received 720mg of subcutaneous nivolumab+rhuph 20, and patients in part B received 720mg of subcutaneous nivolumab, 960mg of subcutaneous nivolumab+rhuph 20, or 960mg of subcutaneous nivolumab. For circulating 2+, patients in part a and part B received 480mg of IV nivolumab every 4 weeks (Q4W). Patients still undergoing the study were converted to part C, i.e., subcutaneous nivolumab 1200mg+rhuph20, until the end of therapy. In part D, the patient received nivolumab 1200mg+rHuPH20 Q4W from under the scalp. Subcutaneous injections are single injections into the abdomen or thigh.

Patient characteristics vary with age, weight, tumor type, and previous treatments. Baseline profiles and disease characteristics for part C and part D are shown in table 60. Tumor types in part a include non-small cell lung cancer (NSCLC), renal Cell Carcinoma (RCC), melanoma (Mel), hepatocellular carcinoma (HCC) and microsatellite-unstable/mismatch repair deficient colorectal cancer (MSI-H/dMMR CRC).

Table 60: baseline basic information/disease characteristics for part C and part D

Nivolumab exposure increased with increasing subcutaneous dose (table 61; fig. 8A-8C). For 960mg and 1200mg of nivolumab+rhuph20, cavg and Ctau were higher than the geometric mean exposure of IV nivolumab 3mg/kg per 2 weeks (Q2W) and Cmax was lower than IV nivolumab 10mg/kg Q2W. In part C (n=28), 13 (46.4%) patients experienced any grade TRAE, but no new/worsening 3+ grade TRAE or resulted in aborted TRAE/death; 7 (25.0%) reported a class 1 local site response. In part D (n=36), 27 (75.0%) patients experienced any grade of TRAE,4 (11.1%) experienced grade 3/4 TRAE,2 (5.6%) experienced severe grade 3/4 TRAE, and 1 resulted in discontinuation, and no treatment-related death; 10 (27.8%) reported grade 1 local site reactions. Anti-nivolumab antibodies (abs) were observed with subcutaneous nivolumab, but were not associated with altered PK/safety or neutralizing antibodies. One (4.5%) patient in part a was considered positive for persistent anti-drug antibodies (ADA) (table 62). In part D, only 1 (3.8%) patient had a positive ADA titer of 8 titer units. No neutralizing ADA was detected and no evidence of altered PK profile and ADA production was present. There is also no correlation of anti-nivolumab antibody production with selected AEs (i.e., bronchospasm, hypersensitivity, infusion-related reactions). Exploratory biomarker data increased cd8+ tumor infiltrating lymphocytes and PD-L1 tumor expression were found in post-treatment biopsies, similar to IV nivolumab.

Table 61: nawushu exposure at doses with rHuPH20

^a For C _max ，n＝22； ^b For C _max ，n＝26；C _tau Concentration at the end of dosing interval

Table 62: NIVO ADA evaluation: portion A, B and D

The safety profile of the exposure associated with the subcutaneous nivolumab+rhuph 20 dose investigated in this study was well tolerated and consistent with IV nivolumab. The data support the evaluation of subcutaneous nivolumab + rHuPH20 in phase 3 studies.

The average duration of subcutaneous nivolumab injections was less than 5 minutes between treatment groups. All patients in part C and part D received complete subcutaneous doses of nivolumab 1200mg+rhuph20 (22,000U) via a single injection.

CD8 (n=23) expression (fig. 9A) and tumor PD-L1 (n=25) (fig. 9B) expression on tumor-infiltrating lymphocytes (TIL) of paired biopsies before and during treatment were evaluated by Immunohistochemistry (IHC). Immunohistochemical results for subcutaneously delivered nivolumab showed 5.0% mean change in CD8 TIL expression and 2.8% mean change in PD-L1 tumor expression, indicating similar pharmacodynamic effects in tumor microenvironment compared to historical intravenous delivery of nivolumab.

EXAMPLE 5 analysis of oxidative stress

In this report, factors that promote increased stability of both nivolumab and rHuPH20, which are two very different proteins, were evaluated in their stable active state at widely different levels in one formulation. Three studies were performed to fully understand the effect of formulation composition.

Study 1-in addition to accelerating stability, a number of oxidative stress conditions were investigated for different combinations of metal, peroxide and light. Study 2-investigation observed concentration ranges for formulation protection. Study 3-investigation of additional major packaging components associated with the drug-loaded syringe and wearable device. This study also carefully examined the formulation effect without rHuPH 20.

To evaluate the stability of nivolumab, size Exclusion Chromatography (SEC) was mainly used, and to evaluate the stability of rHuPH20, enzyme activity was measured. PS80 levels and particulates will be measured at selected time points. In this report, it was shown that the addition of DTPA and Met minimizes the risk of rHuPH20 oxidation and maximizes the stability of nivolumab.

Study design

Study 1: stress conditions

Study 1 was set up to determine the effect of different oxidative stress in combination with each other. The three oxidation stresses studied were: light [ L ] (1000 lux at room temperature), metal stress [ M ] (total 1.5ppm, 0.5ppm each of iron, chromium and copper) and peroxide [ P ] (1 mM peroxide). The three stresses were evaluated separately and combined with each other. Preliminary data show that enzyme activity decreases with RT/RL storage, thus light exposure in combination with other stresses [ LP, LM, MPL ] was maintained at 1000 lux and RT for 3 days (3D). Note that: the light exposure group [ L ] of this study is the only condition exposed to 1000 lux at RT for the complete duration of the time point. Nivo showed little HMW increase at 25 ℃ exposure, thus increasing the standard storage condition temperature to 30 ℃. Since the light combined stress conditions were exposed to light at Room Temperature (RT) for 3 days, all conditions began either at RT/dark for 3 days or at 1000 lux and RT for 3 days. The 8 stress conditions in the study were: illumination/RT (1000 lux) [ L ]; metal stress (1.5 ppm iron, chromium and copper) 3D+30 ℃/dark [ M ] at RT/dark; 1mM peroxide 3D+30 ℃/dark [ P ] at RT/dark; light/RT 3 D+peroxide+30 ℃/dark [ PL ]; light/RT 3 D+metal+30deg.C/darkness [ ML ]; peroxide+metal 3D+30 ℃/dark [ MP ] at RT/dark; light/RT 3 D+metal+peroxide+30 ℃/dark [ MPL ]; RT/dark 3D+30 ℃/dark [ control ].

The center point formulation consisted of: 120mg/mL Nivo, 20mM histidine (pH 6.0), 250mM sucrose, 0.05% w/v polysorbate 80 and 2000U/mL rHuPH20. In this study, the effect of headspace, chelator (DTPA/EDTA) and sacrificial oxidant (Met) was studied as shown in table 63. Table 64 shows the storage conditions and planned time points for this study.

Table 63: study 1-Experimental conditions for oxidation study of BMS-986298

Formulation preparation	Headspace	DTPA(μM)	EDTA(μM)	Met(mM)
					1	Air-conditioner	50	0	5.0
2	Air-conditioner	0	0	0.0
					3	Air-conditioner	0	0	5.0
4	Air-conditioner	50	0	0
					5	Air-conditioner	0	100	5.0
6	Nitrogen and nitrogen	50	0	5.0
					7	Nitrogen and nitrogen	0	0	0

(120 mg/mL BMS-986298 in 20mM histidine (pH 6.0), 250mM sucrose, 0.05% (w/v) polysorbate 80 and 2,000U/mL rHuPH20 were equally present in all formulations)

Table 64: study 1-time Point of Oxidation study

X was withdrawn but not tested. Sample analysis is defined in Table 65

In this report, attention was paid to how the stability of nivolumab (Nivo) was affected by determining different parameters by Size Exclusion Chromatography (SEC). Due to the low throughput of the method, the evaluation of rHuPH20 stability was performed only at selected time points. In addition, the selected samples were chosen for evaluation of the stability of PS80, PS80 being a key excipient added to provide stability of the pharmaceutical product. These results were used to predict how selected conditions affected stability of both Nivo and rHuPH20 under different oxidative stress conditions.

Table 65: study 1 analysis volume

* Subjecting the selected sample to

Study 2: formulation composition differences

Study 2 was set to determine the range of benefits observed for formulation composition. From the point of view of oxidative stress, study 1 defines the conditions to include MPL and RT/RL. Both stress conditions were studied, as well as the oxidative stress of the control. Formulation conditions expected to have oxidative benefits were studied under both oxidative and thermal stress (5 ℃, 25 ℃ and 35 ℃). All formulations were studied using thermal stress conditions to evaluate stability.

Oxidative stress conditions:

MPL: combinations of all three stresses: the standard metal [ M ] (total 1.5ppm, 0.5ppm each of iron, chromium and copper) and the standard peroxide [ P ] (1 mM peroxide) were added at room temperature/room illumination [ L ] (1000 lux) for 3 days. Metals and peroxides are marked at T0 (initial time point). After 3 days at room temperature/room illumination, the samples were removed and stored at 30 ℃ in the dark for the remainder of the time point.

RT30: control of MPL stress conditions. At room temperature/dark for 3 days, followed by 30 ℃ per second at the remainder of the time point in the dark

RT/RL: at room temperature/room illumination (1000 lux) for the complete duration of the study.

The thermal stress conditions include (i) 5 ℃/light shielding; (ii) 25 ℃ away from light (also used as a control for RT/RL conditions); and (iii) 35 ℃/light shielding.

The formulation was chosen to show that the formulation is stable in the following formulation composition: (i) pH:5.2-6.8His; (ii) histidine: 10-100mM (Alt: succinate); (iii) DTPA 10-200. Mu.M (Alt: 100. Mu.M EDTA); (iv) Met:1-20mM (Alt: 10mM Trp); (v) rHuPH20:0-5,000U/mL; (vi) PS80:0.01-0.1% w/v (Alt: PS 20.05% w/v and poloxamer 0.2 mg/mL); (vii) sugar: 10-400mM sucrose (Alt: 10% sorbitol and trehalose); (viii) proteins: 100-175mg/mL. For all these conditions, the center point formulation consisted of: 120mg/mL Nivo, 20mM histidine (pH 6.0), 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% polysorbate 80 and 2,000U/mL rHuPH20, and tested in vials.

Conditions studied for both thermal and oxidative stress conditions are shown in table 66. Note that there are multiple independent formulation formulations in the design for the center point condition and the condition with 50 μm DTPA and no Met. Screening included small excipient characterization DOE, which investigated whether there was any combined effect between pH, DTPA and Met concentrations. In addition to having a nitrogen headspace, differences in DTPA, met and enzyme levels were studied under these conditions, with oxidation being more likely to be affected by changes in these formulations. Alternative excipients are added to understand how they behave with respect to the proposed excipients. As an alternative to Met, trp was added and EDTA was studied as an alternative to DTPA. Higher levels of these alternative excipients were investigated to ensure no problems with respect to the performance of the alternative excipients relative to the proposed excipients. Independent replicates of the selected formulations (formulations 21 and 29 with DTPA only and formulations 15 and 28-center point) were included in the study.

The formulation conditions for the thermal stress survey alone are listed in table 67. The factors that are unlikely to affect oxidation are varied here. These factors are: protein concentration, use of alternative buffers, buffer strength, alternative sugar and sugar concentration, alternative surfactants and varying surfactant levels. The extremum of buffer pH was also studied here. For all these conditions, the formulation included 50. Mu.M DTPA, 5mM Met, and 2,000U/mL rHuPH20.

Stresses and time points corresponding to the oxidation stress conditions are tabulated in table 68. The time points corresponding to the thermal stress conditions are tabulated in table 69.

Table 66: study 2-Experimental conditions for oxidation and thermal stress of BMS-986298

* Multiple independent formulation formulations.

(120 mg/mL BMS-986298 in 20mM histidine, 250mM sucrose and 0.05% (w/v) polysorbate 80 equally present in all formulations)

Table 67: study 2-Experimental conditions of thermal stress only for BMS-986298

* The formulations 15 and 28 in table 66 above were formulated as multiple independent formulations (also tested for thermal stress). This formulation also confirms comparable performance of the two DS sources.

Formulations were performed using different DS sources without sugar.

For this formulation, no sugar was later found to be added. These results were omitted here, but both sugars were covered in study 3.

(50. Mu.M DTPA, 5mM Met and 2,000U/mL rHuPH20 were equally present in all formulations)

Table 68: study 2-time Point of Oxidation study

Sample analysis is defined in table 70. Note that. 1M and 3M samples of MFI where planned but may not be operational due to personnel configuration restrictions during the COVID. * Selectable testing

Table 69: study 2-time Point of thermal study

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Sample analysis is defined in table 70. Note that. A 6M sample of MFI, where planned but may not be operational due to personnel configuration restrictions during the covd. * Selectable testing

In this study, attention was paid to how different parameters influence the stability of BMS-986298 or nivolumab, determined mainly by Size Exclusion Chromatography (SEC). Due to the low throughput of the enzymatic activity method, the evaluation of rHuPH20 stability was performed only at selected time points. In addition, the samples selected were chosen to evaluate the stability of PS80, PS80 being a key excipient added to provide stability of the pharmaceutical product. CE-SDS and iCE were tested on selected samples to understand the formation of low molecular weight species and charge variants. These results were used to predict how selected conditions affected stability of both Nivo and rHuPH20 under different oxidative stress conditions.

Table 70: study 2 analysis volume

* Run on selected samples

Study 3: formulation composition differences between PFS and wearable device

Study 3 was set up similarly to study 2, but the conditions focused on the use of the drug-loaded syringe and wearable device. Similar to study 2, the worst case from the oxidative stress conditions included those from study 1 (MPL). Room temperature/room lighting conditions are removed because such conditions are not likely to be encountered in real life and they impart high stress to both proteins and enzymes. Similar to study 2, standard thermal stress conditions were studied, except for the oxidation stress. For both oxidative and thermal stress conditions, formulation conditions that may affect oxidation were studied. All formulations were tested under standard heat stability conditions.

The oxidation stress conditions include:

RT30: control of MPL stress conditions. The remainder of this time point was continued at 30 ℃/dark after 3 days at room temperature/dark.

The thermal stress conditions include (i) 5 ℃/light shielding; (ii) 25 ℃/light shielding; and (iii) 35 ℃/light shielding.

Using the data from study 2, the formulation concentration range desired for investigation was narrowed to attempt to find the optimal range for stabilization. The formulations were chosen to show that the formulations were stable in these formulation compositions: (i) pH:5.2-6.5His; (ii) histidine: 15-100mM; (iii) DTPA 10-200. Mu.M (Alt: 100. Mu.M EDTA); (iv) Met:1-20mM (Alt: 10mM Trp); (v) rHuPH20:0-10,000U/mL; (vi) PS80:0.01-0.1% w/v (Alt: PS20 and poloxamer 0.2 mg/mL); (vii) sugar: 10-400mM sucrose (Alt: 10% sorbitol and trehalose); (viii) proteins: 100-200mg/mL; and (ix) vials and PFS and patch pumps.

For all these conditions, the center point formulation consisted of: 20mM histidine (pH 6.0), 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v polysorbate 80 and 2,000U/mL rHuPH20. The protein concentration of the sample in the vial was 120mg/mL. The concentration of the Nawuzumab protein in the sample in PFS was 150mg/mL. This study included the various conditions studied in study 2, but without enzyme. Enzyme-induced effects were expected to be minimal, so confirmatory studies were conducted and disposable (one-off) conditions were tested at the center point.

Conditions studied for both thermal and oxidative stress conditions are shown in table 71. Included herein are the conditions added to cover the PFS and the conditions used with respect to the PFS (silicone oil and tungsten plus standard). DTPA, met and enzyme levels were also studied here, where oxidation was more likely to be affected by differences in the composition of these formulations.

The formulation conditions for the thermal stress survey alone are listed in table 72. The factors that are unlikely to affect oxidation are varied here. These factors are: protein concentration, buffer strength and enzyme level. For all these conditions, the formulation included 50 μm DTPA and 5mM Met.

The stress and time points corresponding to the oxidation stress conditions are tabulated in table 73. The time points corresponding to the thermal stress conditions are tabulated in table 74.

Table 71: study 3-Experimental conditions for oxidation and thermal stress of BMS-986298

20mM histidine (pH 6.0), 250mM sucrose and 0.05% (w/v) polysorbate 80 were equally present in all formulations.

Table 72: study 3-Experimental conditions of thermal stress only for BMS-986298

( 50uM DTPA and 5mM Met were equally present in all formulations. Are filled into vials )

Table 73: study 3-time Point of Oxidation study

Sample analysis is defined in table 75. * Optionally testing.

Table 74: study 3-time Point of thermal study

Sample analysis is defined in table 75. * Optionally testing.

In this study, attention was paid to how the stability of BMS-986298 or Nivo is affected by different parameters determined mainly by Size Exclusion Chromatography (SEC). The confirmatory MFI samples were run only on 5 ℃ and 25 ℃ 6M samples.

Table 75: study 3-analytical volume

* Run on selected samples

Materials and methods

Material

Details of the materials used in the study are provided in table 76.

Table 76: material information

Sample preparation

Thawing

Drug Substances (DS) BMS-986298 and rHuPH20 were stored frozen. The DS flasks were thawed at room temperature in the absence of light. Once thawed, the DS bottles were gently mixed to ensure homogeneity. The DS is stored at 5℃until use, and any remaining portion is frozen again after use. The DS used in the formulation study was both DTPA and PS80 free.

For study 1 formulation, DP samples were prepared using bulk DS material BMS-986298 (170 mg/mL) by adding the following stock solutions: (i) 10mg/mL rHuPH20 DS (112 kU/mg rHuPh 20); (ii) 5% polysorbate 80 (100 x); (iii) 2.5mM DTPA (50×); (iv) 5mM EDTA (50X); (v) 250mM methionine (50×); (vi) 20mM histidine, 250mM sucrose, pH 6.0; (vii) 50mM hydrogen peroxide (50 x); (viii) 50ppm of chromium (III) chloride hexahydrate, 50ppm of copper (II) nitrate trihydrate and 50ppm of iron (II) ammonium sulfate (100X). The DS starting composition comprises 20mM histidine and 250mM sucrose, pH 6.0.

For each formulation, samples were prepared with no labeling, with peroxide only, metal only, and metal and peroxide. The metal mixture was prepared immediately prior to the addition of the label. All formulations were filtered after gentle mixing using an acrodisk syringe filter with 0.2 μm Supor membrane. Experiments are recorded in manual A0F 7F-090.

For study 2 formulation, DP samples were prepared by 4 methods. First, by using bulk DS material BMS-986298 (170 mg/mL) added directly, no further preparation-no buffer exchange. Formulation was performed by adding 0.2 μm filtered stock solution to the target concentration. The stock solution includes: 2M sucrose; 495mM histidine, pH6.0;500mM histidine, pH 5.2;500mM histidine, pH 5.5;211mM histidine, pH 6.5;262.5mM histidine, pH 6.8;5% PS80;5mM EDTA;2.5mM DTPA;250mM Met;5% PS80;20mg/mL poloxamer; 1400mM succinate; 80% sorbitol; 40% trehalose; 54mM tryptophan; and water.

This method was used for formulation composition, where DS pH was 6.0 and sucrose was at 250mM level. The DS initial composition is: 20mM histidine, 250mM sucrose, pH 6.0.

CM3: the buffer was exchanged using an automated device, wherein the concentration was performed using a pressurized 10kDa filter, and the target buffer was added to the target buffer exchange composition. Two input DS streams are used: (i) DS in 170mg/mL Nivo stock (this is used as is; DS starting composition: 20mM histidine, 250mM sucrose, pH 6.0); and (ii) DS in 10mg/mL Nivo stock (this material was harvested during the virus filtration step prior to sugar addition; this DS stock uses samples with sugar composition changes; appropriate sugar was added to DS to target composition, then concentrated using a centricon filter; concentrated using a 30kDa filter).

Formulations requiring such buffer exchange have small sample requirements (for only 1 formulation, which requires unique pH or sugar levels). After buffer exchange, the same stock solution was used for formulation as described above. Tangential Flow Filtration (TFF) uses a 30kDa filter and a buffer exchange of 8 dialysis volumes (dianolume). TFF was run twice, once at pH 5.5 and once at pH 6.5. This is desirable because of the formulation with both pH values. After buffer exchange, the same stock solution was used for formulation as in step 1 above. Formulation 38, which is a sugarless formulation, was prepared using dialysis. The solution is initially a sugarless formulation, and then its buffer is exchanged to the target buffer pH/composition. The concentration was then adjusted by centrifugation through a 10kDa membrane. After buffer exchange, the same stock solution was used for formulation as in step 1 above.

For MPL conditions, stock solutions were labeled with metal by producing 50ppm chromium (III) chloride hexahydrate, 50ppm copper (II) nitrate trihydrate, and 50ppm iron (II) ammonium sulfate. Similarly, a 50mM hydrogen peroxide solution was prepared to label the peroxide.

All formulations were filtered after gentle mixing using an acrodisk syringe filter with 0.2 μm Supor membrane.

The TFF buffer was run on an excipient assay to confirm excipient levels. The analysis found that the CM3 buffer exchange with sorbitol was free of added sorbitol and trehalose was also unintentionally lost. Thus, the results from these two samples (formulations 43 and 44) were omitted from the analysis. These conditions were included in study 3 but without enzyme.

For study 3 formulations, DP samples were prepared by direct addition, buffer exchange by CM3, or buffer exchange with TFF. The preparation method was similar to that of study 2. The main difference between study 2 and study 3 is that the DS stock was a different batch of material and one of the metals used for labelling in study 3 was copper (II) chloride, whereas in study 2 was copper (II) nitrate trihydrate.

The patch pump core pack is filled with a needle and placed in a steady state with the vial and syringe. Samples were withdrawn at the appropriate withdrawal time and frozen until the Smart Dose patch pump reached. The solution is delivered from a core pack using the patch pump. The liquid was withdrawn using a needle for the control sample.

Filling

The formulated and filtered DP was filled into depyrogenated 3cc vials (Schott type 1 glass vials) or BD Neopack 2.25mL syringes and stoppered with 13mm Daikyo stoppers (D-21-7S, fluotec coated serum) or BD1-3mL plungers treated with high pressure steam. The vials were screwed on and placed upright and stable in the vertical position. The exception was at exposure Yu Guangshi, at which time the vial was placed horizontally. The syringe is stored in a horizontal position.

Analysis

Samples were withdrawn and immediately frozen until analysis time, with the exception of microparticle analysis (MFI); it was kept at 5 ℃ until analysis.

SEC

DP samples were analyzed by SEC-HPLC (MTD-10789) for quantitative analysis of HMW and LMW species. Samples were analyzed using a TSK gel Super SW 3000 column (TOSOH 1875) at a flow rate of 0.5mL/min and run time of 35min. The mobile phase was 100mM phosphate and 100mM sodium sulfate, pH 6.8. A 50 μg loading of Nivo sample was injected per run. Peaks were detected by UV absorbance at 280nm, and the area and retention time of each peak were measured. The area percentages of monomer, HMW and LMW species were calculated and reported.

Enzyme Activity

The rHuPH20 activity was measured using a plate-based turbidity assay, method CTL-10028 in DCA. Hyaluronidase potency assays are based on the formation of insoluble precipitates when Hyaluronic Acid (HA) is combined with acidified serum. The precipitate resulted in a turbid solution that could be measured at 640 nm. The efficacy (or activity) of hyaluronidase is measured directly by: the enzyme was incubated with HA substrate for 30 minutes, undigested HA was precipitated with acidified serum and turbidity was compared to a reference standard curve. PS80 analysis

PS80 levels were measured with a mass spectrometer at 30 ℃ using a Waters Oasis Max column with 0.1% formic acid and 0.1% formic acid in 5mM ammonium formate/acetonitrile at a flow rate of 1 mL/min.

Microparticles

Microparticle levels were tested using microfluidic imaging (MFI). 1.1mL of each sample was filled into separate glass vials for MFI analysis. The test samples were run as water samples between analyses. The samples were tested at a purge of 0.15mL and an analytical volume of 0.43 mL.

Charge variants determined by imaging capillary isoelectric focusing (iCIEF)

The charge variants were analyzed by iCIEF using an imaging isoelectric focusing system called iCE3 (MTD-10788). Samples mixed with appropriate pI labels, ampholytes and other additives were injected by an autosampler into Fluorocarbon (FC) coated capillary cartridges. After high voltage pre-focusing and focusing, sample migration is captured by a UV detector and a UV light absorption image is taken by a CCD camera. The area percentages of the main peak, acidic variant and basic variant were calculated and reported.

CE-SDS

The relative purity percentages of Nivo were determined by CE-SDS using a LabChip GX II caliper system under non-reducing conditions. Samples were denatured and prepared in the presence of Sodium Dodecyl Sulfate (SDS), a detergent that coats the protein, providing a negative charge to effectively mask the natural charge of the protein. Each sample was aspirated onto the chip, mixed with dye and separated by electrophoresis. A separate decolorization step is then performed on the chip. An optical element within the instrument detects the fluorescent signal of each sample. The protein species are separated based on size and electrophoretic mobility. The relative area percent of each peak was calculated and the relative purity percent (% main peak) was reported.

Results-study 1

Size exclusion chromatography

Size exclusion chromatography is a major tool for monitoring the stability of nivolumab under the various stress conditions studied.

To investigate the effect of the nitrogen headspace, two formulations with a nitrogen headspace were investigated. Formulations with both 50 μm DTPA and 5mM Met (formulation 1-air, formulation 6-nitrogen) and formulations without either DTPA or Met (formulation 2-air, formulation 7-nitrogen). The HMW variation is plotted in figure 10. For formulations with both DTPA and Met, the headspace effect was minimal for all conditions except for RT/light conditions with continuous light exposure (fig. 10). In the formulation without DTPA and without met, there was a nitrogen headspace present benefit (fig. 10). In the case of both DTPA and Met addition, the control of oxidation is good, so a nitrogen headspace is not required.

The effect of various formulations on the formation of HMW species of Nivo after exposure to various stress conditions is shown in fig. 11. The stress conditions include (from left to right): 1. ) The exposure was controlled for 3 days at room temperature/darkness (3 day light exposure for L conditions) +30 ℃ thermal stress (thermal stress for all conditions except RT/light); 2. ) Metal-iron, chromium and copper were each 0.5ppm (M conditions) +illumination (3 days-L conditions at 1000 lux and room temperature) +30 ℃ thermal stress; 3. ) M+1mM peroxide (P conditions) +L+30deg.C thermal stress; 4. ) M+l+30 ℃ thermal stress; 5. ) Thermal stress at m+30 ℃;6. ) P+l+30 ℃ thermal stress; 7. ) P+30 ℃ thermal stress; 8. ) Room temperature/room lighting (no additional thermal stress).

Of all stress conditions, formulation 2 without DTPA and without Met had the highest HMW formation rate, followed by formulation 3 without DTPA and with 5mM Met. Formulation 5 with 100mM EDTA and 5mM Met, followed by formulation 4 with 50. Mu.M DTPA and no Met. The best formulation is formulation 1 with both 50 μm DTPA and 5mM Met. This behavior was observed under all stress conditions, but the differences were best observed by looking at MPL (combined stress conditions with 30 ℃ thermal stress). A graph of this MPL stress condition for all formulation conditions is shown in fig. 12.

Careful examination of MPL stress conditions in fig. 12 showed benefits with both 50 μm DTPA and 5mM Met (formulation 1-air and formulation 6-nitrogen). Having two antioxidant stabilizations Nivo in the formulation, such that the replacement of the headspace with nitrogen did not show improved stabilization (formulation 1 and formulation 6 were fully overlapped). Only with DTPA (formulation 4:50. Mu.M DTPA and 0mM Met) outweighed by the larger amounts of EDTA and Met (formulation 5:100. Mu.M EDTA and 5mM Met). The highest HMW formation was observed in the air headspace without DTPA and without Met (formulation 2:0. Mu.M DTPA and 0mM Met), followed by the same formulation but in the nitrogen headspace (formulation 7) and with only Met (formulation 3:0. Mu.M DTPA and 5mM Met).

Another way to check these data is by looking at the formulation compositions and how they behave in stress conditions, as shown in fig. 13. Benefits of both DTPA and Met can be observed. This way of examining the data shows that RT/light conditions are worst case and that from a stress alone perspective, 3 days of light and/or metal results in the highest% HMW formation and the effect of peroxide is diminished. For RT/light conditions, the benefit of nitrogen headspace is very clear. For the sample without DTPA, without Met, the nitrogen headspace reduced the HMW% by about 0.5% in all stress conditions. No significant improvement in nitrogen headspace was observed for the formulations with both DTPA and Met. The HMW% of 100 μm EDTA and 5mM Met is greater than 50 μm DTPA and 5mM Met, but the magnitude of the effect may depend on the exact stressor.

To provide statistical evaluation, various linear models were explored. The absence of pure error estimates, design imbalance, and complex formulation-stress dynamics precludes attempts to incorporate all factors in a single combined model. Nitrogen and EDTA samples were excluded from the analysis. The remaining chelator-methionine observations are shown in fig. 14 and provide an image motivation for the analysis described below. Although included in the statistical model, RT/room lighting conditions have been removed from the following figures, as such extreme stress conditions are less relevant to the intended use case.

The potential for chelator-stress interactions is evident for this reductive dataset. However, in DTPA-only samples, the stress effect appears to be additive. This suggests that the primary effect statistical model is predictable and can be used to compare DTPA with/without methionine. Residual variance differences are possible based on the presence or absence of DTPA.

Based on the presented analysis, a comparison of DTPA with or without 5mM methionine using an air headspace was carefully examined. Using DTPA-only samples under air, the primary linear model of the incorporation of methionine and stress estimated that methionine addition had a marginally significant average benefit in HMW% at 4 weeks (difference 0.0375%, p about 0.0796) and a statistically significant average improvement in HMW% in all stress conditions at 12 weeks (difference 0.125%, p about 0.0016) (data not shown).

Polysorbate 80

The subset of samples were tested to understand the stability of polysorbate, a key surfactant to prevent Nivo from forming particulates. The PS80 levels of the samples tested are shown in table 77. PS80 is indeed oxidized by metals, peroxides and light. Degradation is reduced by the addition of chelating agents (DTPA or EDTA). After storage without chelating agent, PS80 levels decreased significantly. This drop was observed after 2 months even when stored at 5 ℃. After 1 month storage at 30 ℃ and under metal and peroxide stress (wherein 3 days are at room temperature/room illumination), the sample without chelating agent falls to less than 0.1mg/mL (0.5 mg/mL target from the start). The nitrogen headspace slightly protected PS80 (0.08 mg/mL under nitrogen versus 0.02mg/mL under air).

Table 77: study 1-PS 80 levels at 5 ℃ for 2 months and 3 days later 1 month under light + metal + peroxide +30 ℃/dark [ MPL ], starting from 0.5mg/mL of initial PS 80.

rHuPH20 enzyme Activity

To assess the stability of rHuPH20, the enzyme activity of a bounded set of samples was measured. The results of the enzyme activity measurement are shown in FIG. 15. The enzyme levels of the samples at 30 ℃/dark and MPL stress (metal and peroxide stress, where the first 3 days were at room temperature/room illumination) are tabulated in fig. 15 (middle panel). The formulation with both DTPA and Met (formulation 1) outperformed the formulation with DTPA alone, and the formulation with DTPA alone outperformed the formulation with Met alone. Formulations without them (neither DTPA nor methionine) had the greatest decrease in enzyme activity.

The samples exposed to 3 days RT/darkness and 30 ℃/darkness after that in fig. 15 (left panel) showed similar behavior, with the formulations with DTPA and Met and the formulations with DTPA alone outperformed the formulations with Met alone and the formulations without DPTA and Met.

For samples exposed to RT/RL, the rHuH20 enzyme activity showed different behavior, FIG. 15 (right panel). For these conditions, all formulations had unreported (low) levels of rHuPH20 activity for all 3M samples, with the exception of samples with DTPA and Met kept at nitrogen headspace. No formulation differences were observed with continuous exposure to indoor lighting and air in the headspace. These results indicate that the combination of headspace nitrogen with DTPA and Met helps to protect the enzyme under continuous exposure to room temperature/room illumination.

Particle formation

For all stress conditions and formulations, particle formation was evaluated at two week time points. For all samples, the level of macroparticles was low (less than 32 particles/mL) for particles of 25 μm. For all samples, the particle size of the.gtoreq.10 μm particles was below 164 particles/mL. The sample with the highest particle count is under room temperature/room illumination, but the particle count is much lower than the USP <788> specification. No particulate problem was observed during this study.

The stability of the formulation was monitored by three key components: stability of Nivo as determined by SEC, stability of rHuPH20 as determined by monitoring enzyme activity, and stability of PS80 as determined by measuring PS80 levels. For each of these quality attributes, formulation performance and ranking, ordered from best formulation to worst (top to bottom) formulation, are tabulated in table 78.

Nivo stability as determined by SEC showed an increase in HMW in response to oxidative stress. The increase in HMW is prevented by the presence of antioxidants under various combined stresses (metal, peroxide and light). The combination of Met with DTPA showed higher stability than DTPA alone, which outperforms Met alone, which outperforms the formulation without DTPA nor Met.

rHuPH20 enzyme activity decreases in response to oxidative stress. In particular, the decrease due to the optical stress is remarkable. Formulation composition benefits can be observed for a combination of metal, peroxide and photo-stress (3 day) conditions. Preservation of enzymatic activity is optimal in the presence of both Met and DTPA. There is a measurable decrease in enzyme activity relative to DTPA alone, which has a measurable decrease relative to Met alone, which has an activity relative to neither Met nor DTPA alone.

PS80 protection was observed for conditions with chelator (DTPA). Met does not protect PS80 from degradation.

Table 78: study 1-quality attributes studied that enable the differentiation of the assignment of articles and ranking of these formulations

Key results from investigating various combinations of peroxide, metal and light exposure and adding 30 ℃ thermal stress and various formulations showed that there was benefit in formulations with both 50 μm DTPA and 5mM Met. The use of only one of these antioxidants is less stable than having both a chelating agent and a sacrificial antioxidant.

Results-study 2

The formulations tabulated in tables 68 and 69 were analyzed. In this study, the aim was to understand the factors and scope in which the benefits of this formulation can be observed. The factors and ranges explored are: pH:5.2-6.8His; histidine: 10-100mM (Alt: succinate); DTPA 10-200. Mu.M (Alt: 100. Mu.M EDTA); met:1-20mM (Alt: 10mM Trp); rHuPH20:0-5,000U/mL; PS80:0.01-0.1% w/v (Alt: PS20 and poloxamer 0.2 mg/mL); sugar: 10-400mM sucrose; and (3) protein: 100-175mg/mL. This resulted in 49 different formulations to be studied here. The pH, DTPA and Met levels are co-varying. Formulations with excipient ranges max and min were varied for each single variable, with all other factors at the target composition. The target composition is as follows: 120mg/mL Nivo in 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/v PS80 (pH 6.0) with an air headspace. Note that in the design for the center point condition and the condition with 50 μm DTPA and no Met, there are multiple independent formulation formulations. The distribution of the formulations studied is shown in fig. 16A-16F.

Similar to the formulation range studied at the target composition, alternative excipients were also studied: succinate, 100. Mu.M EDTA, 10mM Trp, 0.05% w/v PS20, poloxamer 0.2mg/mL, 10% sorbitol and trehalose, other excipients at the target composition.

Size exclusion chromatography

Size exclusion chromatography is a major tool for monitoring the stability of nivolumab.

Reproducibility of

Multiple and independent formulation formulations were included in the design for the center point condition and the condition with 50 μm DTPA and no Met. The final HMWs of these formulations, as determined by SEC at stress and time points, are tabulated in table 79.

Table 79: study 2-high molecular weight species-centre and 0Met formulation determined by SEC for the last time point in each stress condition studied for duplicate samples. Center formulation composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0.0Met composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 0.05% w/v PS80, pH 6.0.

Enzyme level

In this study, a range of enzyme levels between 0 and 5,000U/mL was studied. For this stress condition, the final HMW, as determined by SEC for various enzyme levels, is tabulated in table 80 for the final time point. The formulations consisted of enzymes with different levels, 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0. In all stress conditions studied, enzyme levels had no effect on the formation of HMW species.

Table 80: study 2-high molecular weight species determined by SEC for the last time point in each stress condition studied for various enzyme levels. The preparation comprises the following components: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% PS80, pH 6.0.

Alternative excipients

In this study, alternative excipients were studied with a center composition for all other excipients. As an alternative surfactant to 0.05% w/v PS80, 0.05% w/v PS20 and 0.2mg/mL poloxamer were studied. As an alternative buffer to 20mM histidine, 20mM succinate was studied. Surfactants and buffer substitutes are unlikely to have a large impact on protection against oxidation, and therefore the performance of these substitutes was investigated under thermal stress conditions. For this stress condition (target formulation composition 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50 μm DTPA, 5mM Met, 2,000u/mL rHuPH20, 0.05% w/v PS80, pH 6.0) adjacent to the center point condition, the final HMW determined by SEC using these alternative excipients is tabulated in table 81 for the final time point. As an alternative excipient PS80 can be replaced by PS20 or poloxamers with little impact on stability. Formulations with histidine as buffer are essential for Nivo stability and cannot be replaced by succinate. The HMW species increased significantly when succinate buffer was used in all thermal stress conditions studied (5 ℃, 25 ℃ and 35 ℃).

Table 81: study 2-high molecular weight species determined by SEC for the last time point in each stress condition studied for the alternative excipient. Center point composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

As an alternative chelator to 50. Mu.M DTPA, 100. Mu.M EDTA was studied. DTPA is a better chelator, so higher levels of EDTA were studied here. Similarly, 10mM tryptophan was studied as an alternative sacrificial agent to 5mM methionine. Since the addition of chelating agents and sacrificial oxidants may affect protection against oxidation, these chelating agents and sacrificial oxidants are investigated under both thermal and oxidative stress conditions. For this stress condition (center point formulation composition 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50 μm DTPA, 5mM Met, 2,000u/mL rHuPH20, 0.05% ps80, ph 6.0) adjacent to the center point conditions and corresponding controls, the final HMWs determined by SEC using these alternative excipients are tabulated in table 82 for the final time point.

Table 82: study 2 alternative excipients for DTPA and Met in each of the stress conditions studied, high molecular weight species determined by SEC for the last time point. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

The tabulated HMW% values after 3 months under MPL stress clearly show the unique benefit of DTPA and are more excellent chelators than EDTA. The% HMW observed after 3 months under MPL stress was 1.6% compared to 2.1% HMW. Evaluation of chelator alone (without Met) further illustrates the benefits of DTPA versus EDTA (1.8% hmw for DTPA versus 3.7% hmw for EDTA after MPL stress at 3M). Similarly, the data shows that Met is better than Trp. The observed HMW was 1.9% for Met versus 2.4% for Trp after 1M at RT/RL and 1.5% for Met versus 1.9% HMW for Trp after 3M MPL. Control of% increase in HMW was optimal when formulated with 5mM Met and 50. Mu.M DTPA. These results are consistent with study 1.

Level of DTPA

The range of DTPA levels from 0-200 μm was studied with the following center point formulation composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0. For this stress condition, the final HMW, as determined by SEC at various Met levels, is tabulated in table 83 for the final time point. In accelerated thermal conditions (25 ℃ and 35 ℃) and MPL oxidation conditions, there is an effect of DTPA with 0 μm; however, between 10-200 μm concentrations, no convincing evidence of concentration-dependent DTPA effect was observed. This behavior is consistent between stress conditions over time, as shown in fig. 17.

Table 83: study 2 for various DTPA levels, in each of the stress conditions studied, for the last time point, the high molecular weight species was determined by SEC. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 5mM Met, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

Methionine levels

The range of methionine (Met) levels from 0-20mM Met was studied with the following center point formulation composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0. For this stress condition, the final HMW, as determined by SEC at various Met levels, is tabulated in table 84 for the final time point. Changes in Met levels did not result in significant effects on thermal conditions (5 ℃, 25 ℃, 35 ℃ and RT/30 ℃ control). The benefits of high levels of Met were observed when combined with DTPA over 1 month under oxidative stress conditions of RT/RL and 3 months under oxidative stress conditions of MPL. The benefits are minimal due to the greater impact of adding DTPA to HMW formation in all of these formulations.

Table 84: study 2 for various Met levels, in each of the stress conditions studied, for the last time point, the high molecular weight species was determined by SEC. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 0.05% w/v PS80, 2000U/mL rHuPH20, pH 6.0.

The time course change in HMW% under each condition is shown in figure 18. Of the various stress conditions, 0 μmdtpa and 0mM Met had the highest HMW levels. The distinction between 0 and 5mM methionine formulation at 0. Mu.M DTPA level is evident, and the addition of Met is beneficial for stability of Nivo. At the target 50 μm DTPA concentration, the effect of Met levels at various stresses is more indistinguishable at the 2 week and 1 month time points. However, at the 3 month and 6 month time points, careful evaluation is required.

Careful evaluation of MPL (metal + peroxide + 30 ℃/dark stress conditions after 3D light) is shown in figure 19. Note that: for formulations with 0mM Met and 50 μm DTPA and formulations with 5mM Met and 50 μm DTPA, there are multiple independent formulations labeled with two and three signs. 0 and 5mM Met formulations at 0. Mu.M DTPA were distinguished at all three time points. At 50 μm DTPA concentration, HMW formation% appears to vary with methionine concentration at two time points of 1 month and 3 months under these MPL conditions. In particular, for MPL stress conditions at the 3 month time point, the benefit of having a higher level of Met was clearly observed. HWM formation at 100 μm and 200 μm DTPA and 5mM Met concentrations was similar to 50 μm DTPA and 5mM Met levels. This is due to the binary effect of DTPA, but there is no convincing evidence of concentration-dependent DTPA effect as discussed above.

pH

The range of pH levels from 5.2 to 6.8 was studied with the following center point formulation composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/vPS. For this stress condition, the final HMWs determined by SEC at various pH levels are tabulated in table 85 for the final time point. Having a higher pH has an effect in all thermal conditions (5 ℃, 25 ℃ and 35 ℃) and oxidative conditions (RT/RL, MPL and RT/30 ℃ control). Between a pH range of 5.2 to 6.5, the formulation has good stability.

Table 85: study 2 for various pH levels, in each of the stress conditions studied, for the last time point, the high molecular weight species was determined by SEC. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80 and 2000U/mL rHuPH20.

DOE evaluation of pH, met and DTPA levels

There are small excipient characterization DOEs that operate on a combination of pH, met and DTPA levels. Formulations in DOE are tabulated in Table 86, all containing 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 2,000U/mL rHuPH20 and 0.05% PS80. For this stress condition, the final HMW, as determined by SEC at various Met levels, will be tabulated for the final time point. Examination of the results showed a clear pH effect in all stress conditions. The results of this part of the study are consistent with the single variability study results described above. For MPL conditions, the main effect is pH, with higher pH leading to higher HMW values. Higher levels of Met present protection and DTPA levels studied here (10-100. Mu.M) did not affect HMW.

Table 86: study 2 DOE for investigating the effect of pH, met and DTPA levels, in each of the stress conditions studied, the high molecular weight species determined by SEC for the last time point. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80 and 2000U/mL rHuPH20.

Effects of histidine levels

The range of histidine levels from 10 to 100mM was studied with the following center point formulation composition: 120mg/mL Nivo, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/vPS, pH 6.0. For this stress condition, the final HMWs determined by SEC at various histidine levels are tabulated in table 87 for the final time points. The data indicate that histidine is not only a buffer in the formulation, but also has protective properties at higher histidine levels, thus increasing protein stability. At low histidine concentrations, protection is minimized, especially below 20mM histidine. The data herein show cliffs below this 20mM histidine level. The exact location of the cliff is unknown. Histidine concentrations ranging from 15mM to 100mM are expected to have comparable and good stability.

Table 87: study 2 for various histidine levels, high molecular weight species were determined by SEC for the last time point in each stress condition studied. The composition comprises: 120mg/mL Nivo, 250mM sucrose, 50. Mu. MDTPA, 5mM Met, 0.05% w/v PS80 and 2000U/mL rHuPH20, pH 6.0

Influence of protein concentration

The protein concentration range studied was 100 to 175mg/mL with a center point formulation composition of 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0. For this stress condition, the final HMWs, determined by SEC at various protein concentrations, are tabulated in table 88 for the final time point. Higher protein concentrations showed higher HMW formation and are consistent with other studies conducted at higher protein concentrations.

Table 88: study 2 for various protein concentrations, in each of the stress conditions studied, for the last time point, the high molecular weight species was determined by SEC. The composition comprises: 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80 and 2000U/mL rHuPH20, pH 6.0

Influence of sucrose concentration

Sucrose concentrations studied ranged from 0 to 400mM with a center point formulation composition of 120mg/mL Nivo, 20mM histidine, 50 μM DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0. For this stress condition, the final HMWs determined by SEC at various sucrose concentrations are tabulated in table 89 for the final time points. Low sucrose levels have lower stability. The elevated HMW values are independent of the range of 10-400mM sucrose.

Table 89: study 2 for various sucrose concentrations, the high molecular weight species was determined by SEC for the last time point in each stress condition studied. The composition comprises: 120mg/mL Nivo, 20mM histidine, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80 and 2000U/mL rHuPH20, pH 6.0

Effects of PS80 concentration

PS80 concentrations studied ranged from 0 to 0.10w/v PS80 with a center point formulation composition of 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, pH 6.0. For this stress condition, the final HMWs determined by SEC at various PS80 concentrations are tabulated in table 90 for the final time point. The observed HMW values were constant over the range of formulation compositions studied.

Table 90: study 2 for various PS80 concentrations, in each of the stress conditions studied, the high molecular weight species was determined by SEC for the last time point. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met and 2000U/mL rHuPH20, pH 6.0

Influence of nitrogen headspace

To understand the benefits of the formulation-the nitrogen headspace was used to determine its effect on HMW during oxidative as well as thermal stress. The two formulations studied were the target center point formulation conditions: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0; and conditions without either DTPA or Met: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0. For this stress condition, the final HMW, as determined by SEC, is tabulated in table 91 for both formulations and the nitrogen and air headspace for the final time point.

Table 91: study 2 for 2 formulations with and without nitrogen headspace, high molecular weight species were determined by SEC for the last time point in each stress condition studied. The center composition was 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80 and 2000U/mL rHuPH20, pH 6.0.0DTPA 0Met refers to the removal of both antioxidants (DTPA and Met) from the center point formulation.

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The data show the clear benefit of a center point formulation with both DTPA and Met over a composition without both DPTA and Met. There is a nitrogen headspace benefit in the absence of added antioxidants (sacrificial and chelating agents). An interesting manifestation is a slight difference in the performance of the center point formulation under RT/RL conditions. Here, the nitrogen headspace helps to reduce HMW formation. However, under MPL conditions, the nitrogen headspace does not affect HMW formation in the presence of both the chelating and sacrificial agents. Formulations containing both sacrificial and chelating agents are able to protect against HMW formation from weak light (3 days), peroxide and oxidative stress of metals, compared to the case where neither sacrificial nor chelating agents are present.

Statistical model: final time point for the DTPA concentration formulation at pH 6 50 μm

The statistical model was evaluated for total HMW%. Only statistical models for subsets of stress conditions were developed due to the small/negligible effects observed in most stress conditions. Various hybrid models (not shown) were also explored, assuming a linear model for the HMW% versus time (days). Due to design constraints, at 12 weeks, for the stress conditions, a limited number of degrees of freedom for estimating inter-and intra-formulation variability and HMW% mean square error (RMSE) of about 0.15% to 0.25% average confidence interval are substantially overlapping for the key comparison. For the two key formulation comparisons (above), the results from the first study estimated an average HMW% difference of 0.125%. All analyses were performed using JMP 15.2.0.

Close evaluation of 25 ℃, 35 ℃ and MPL conditions provided a weaknesses to the clear evidence of the beneficial effects of the no-yes (no-yes) DTPA at pH 6 for these three stress conditions at least the longest time point. Furthermore, for the DTPA-free pH 6 formulation, a methionine effect of 0-5mM no-was observed. To simplify the presentation of the results, a statistical model of each of the three stress conditions was developed only at the time point of the last measurement. HMW% RMSE of about 0.15% to 0.25% under various statistical models was observed even when limited to pH 6 only formulations. To further reduce the need for a potentially unsuitable empirical model, only statistical results at 50 μm DTPA are summarized. Model complexity and/or nonlinearity (e.g., due to inclusion of DTPA-free samples) may increase the residual variance of the model for either wrong or under-specified models.

FIG. 20 shows a graph of SEC HMW% versus methionine concentration under stress conditions with three final samples of a smooth curve trend estimate.

For the 35 ℃ 3 month samples, the repeat variability with respect to the residual signal was large. For 25-6 month measurements, only three different values were obtained. For both thermal stress conditions, little or no (lite-or-no) trend is evident. For the combined oxidative stress-MPL conditions, the near-linear trend was most pronounced at 3 months.

A simple linear regression of each condition was estimated to evaluate the intensity of the observed relationship (fig. 21A-21C).

Only the combined oxidative stress conditions (MPL) linear regression was significant (p was about 0.0088). These data demonstrate the significant benefit of increasing methionine concentration in the presence of 50 μm DTPA for this oxidative stress condition. No model lacking the fit was detected (curve model was also estimated with a modified fit, not shown) and in all three regressions the average HMW% RMSE was approximately 0.06% to 0.08%. For a 50. Mu.M DTPA-0mM methionine formulation, the predicted average HMW% at 3 months is 1.73% (95% confidence interval 1.62-1.83); the corresponding 50. Mu.M DTPA-5mM methionine estimate was 1.63% (95% confidence interval 1.55-1.71). The average difference between these estimates was approximately equal to the 0.125% average difference observed between the same two formulations at 3 months under MPL oxidative stress conditions in the first study (above).

Charge variants

Charge variants were evaluated using iCIEF. Detailed studies focused on a subset of samples with the benefits of both antioxidants to evaluate the effect of charge variants on Nivo. The samples tested were 3M time points for MPL oxidative stress conditions and 3M time points for thermal stress at 35 ℃. The results are graphically shown in fig. 22A-22B.

For the condition without antioxidant (none), the% acidic species is highest. Only methionine is less protected than with DTPA alone or both DTPA and methionine. There was no difference between the formulation with DTPA alone and the formulation with both DTPA and Met when stored at 35 ℃. A slight benefit can be observed at 3 months time points at 3 months under MPL stress.

CE-SDS

The clipping (clipped) species of Nivo were measured using CE-SDS. Detailed studies focused on a subset of samples with the benefits of both antioxidants to evaluate the effect of the formulation on prevention of Nivo oxidation. The samples tested were 3M time points and 35 ℃ 3M time points for MPL oxidative stress conditions. The results of the area% of the main peak are tabulated in table 92. No significant difference in main peak area was detected between the samples relative to the initial (T0) samples. No change in LMW species formation was detected in the studied conditions between the studied formulations.

Table 92: study 2 the percentage of the main peak area of CE-SDS as a function of time under MPL conditions and stress at 35 ℃. For formulations with DTPA and Met, for samples with DTPA alone, duplicate samples were prepared. The formulation had a DTPA concentration of 50. Mu.M and a Met concentration of 5mM. All formulations also contained 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, 2,000U/mL rHuPH20, pH 6.0

Enzyme Activity

Enzyme activity was measured to determine the stability of rHuPH20 enzyme. Detailed studies focused on a subset of samples with the benefits of both antioxidants to evaluate the effect of the formulation on protection from enzymatic oxidation (primary degradation mechanism). The samples tested were 3M time points and 35 ℃ 3M time points for MPL oxidative stress conditions. The results are graphically shown in fig. 23A-23B.

Enzymatic degradation is highest for the condition without antioxidant (none). Methionine only is less protected than DTPA only. Having both DTPA and methionine provides optimal protection. The benefits were more pronounced when looking at MPL stress conditions, but a consistent trend was observed between 35 ℃ and MPL conditions. These results are consistent with those observed in study 1 above.

PS80 stability

PS80 stability was measured to determine the stability of key excipient PS 80. Detailed studies focused on a subset of samples with the benefits of both antioxidants to evaluate the effect of the formulation on protection against PS80 oxidation. The samples tested were 3M time points and 35 ℃ 3M time points for MPL oxidative stress conditions. The results are graphically shown in fig. 24A-24B.

Under MPL stress conditions, PS80 degradation was high and PS80 levels dropped to zero for conditions without DTPA (data not shown). For 35 ℃ conditions at the 3M time point, no difference from Met alone can be observed. The kinetics of PS80 degradation were fastest for the antioxidant-free condition (none), followed by methionine-only condition. Both MPL and 35 ℃ stress conditions showed comparable protection of PS80 between the formulation with DTPA alone and the formulation with both DTPA and methionine. From the PS80 stability point of view, a key feature is the inclusion of DTPA. These results are consistent with those observed in study 1.

Formulation latitude and alternative excipients

Study 2 was set up to determine the extent of impact and benefits of formulation composition observed mainly by SEC. The formulations were stressed with thermal stress alone (5 ℃, 25 ℃, 35 ℃) or with thermal stress and oxidative stress (MPL, RT/RL).

Based on this study, the formulation composition ranges that maintain the best stability are as follows: pH:5.2-6.5His; histidine: 15-100mM; DTPA: 10-200. Mu.M (Alt: 100. Mu.M EDTA); met:1-20mM (Alt: 10mM Trp); rHuPH20:0-5,000U/mL; PS80: 0.01-0.1% w/v (Alt: PS 20.05% w/v and poloxamer 0.2 mg/mL); sugar: 10-400mM sucrose; and (3) protein: 100-175mg/mL.

The center point formulation is: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 5mM Met, 50. Mu.M DTPA, 0.05% w/v polysorbate 80, pH 6.0, and 2,000U/mL rHuPH20.

Histidine buffer is critical in formulations, formulations with the same pH using succinate as buffer do not show sufficient stability (table 91). Histidine has a stabilizing effect, which shows increased stability at higher concentrations. In this study, up to 100mM histidine was studied (Table 87), but higher histidine concentrations were also expected to be stable. At low histidine concentrations, stability decreases. In particular, at histidine concentrations of less than 10mM, the% HMW increases. A range between 15-100mM is critical in minimizing HMW species formation.

The pH range is also critical, with higher pH above pH 6.5 showing an increase in HMW with respect to stability (table 85).

Nivo in the absence of sucrose resulted in slightly higher levels of HMW formation (table 89). Sucrose between 10-400mM showed good stability.

PS80 levels between 0 and 0.1% studied here prevented Nivo from HMW formation (Table 90). Alternative surfactants: PS20 (0.05% w/v) and poloxamer (0.2 mg/mL) showed good protection against Nivo against HMW formation (Table 81).

The addition of enzymes ranging between 0 and 5,000U/mL did not affect the stability of Nivo (Table 80).

This report shows the need for two antioxidants. In addition to the chelating agent (DTPA), a sacrificial agent (Met) is also needed. In particular, both antioxidants perform better than their alternative excipients (table 82). To confirm the superiority of the selected excipients, the performance of alternative oxidants has been tested at higher concentrations. Replacement of 5mM Met with 10mM Trp resulted in higher HMW under both RT/RL conditions and under MPL stress conditions. The performance of 50. Mu.M DTPA replaced with 100. Mu.M EDTA under RT/RL conditions was comparable, but showed higher HMW species formation under accelerated heat conditions and under MPL stress conditions. Oxidative protection of the combined formulation does not require a nitrogen headspace to protect against MPL stress (table 91).

DTPA levels between 10-200 μm have shown comparable stability (table 83). DTPA with the lowest concentration of 10 μm is critical, but higher levels of DTPA do not increase the stability of Nivo.

Increases in Met have shown acceptable stability at levels ranging from 1-20mM (Table 84). For the case of Met, a statistical model was constructed and for MPL stress conditions, linear regression was found to be significant (above). This shows that there is a clear benefit with Met and that higher concentrations of Met are advantageous for resisting MPL stress.

Ranking order

The benefits of having two antioxidants were demonstrated by monitoring the following: stability of Nivo (by SEC and iCIEF), stability of rHuPH20 (by looking at enzyme activity), and stability of PS80 (by looking at PS80 levels). This part of the study focused on a subset of samples that required two antioxidants (two compared to only one compared to none). For each of these quality attributes, formulation performance and ranking, ordered from best formulation to worst (top to bottom) formulation, are tabulated in table 93.

Table 93: study 2-quality attributes studied that enable the differentiation of the assignment of articles and ranking of these formulations

Nivo stability as determined by SEC showed an increase in HMW in response to oxidative stress (MPL). The increase in HMW is minimized by the presence of an antioxidant. The combination of Met with DTPA showed higher stability than DTPA alone, which outperforms Met alone, which outperforms the formulation without either DTPA or Met. These results are consistent with study 1.

The charge variants (above) showed that the% increase in acidic species was highest without DTPA and without Met. Having Met only shows a slightly slower rate of increase for both thermal (35 ℃) and oxidative (MPL) stress conditions. Under thermal stress conditions, having DTPA alone is comparable to having both DTPA and Met; however, under MPL conditions, there are minor benefits observed when there are two compared to DTPA alone.

rHuPH20 enzyme activity decreases in response to both thermal and oxidative stress. Formulation composition benefits can be best observed for oxidative stress (MPL). Preservation of enzymatic activity is optimal in the presence of both Met and DTPA. There is a measurable decrease in enzyme activity relative to DTPA alone, which has a measurable decrease relative to Met alone, which has a measurable decrease relative to neither Met nor DTPA. This behavior is consistent for thermal stress (35 ℃) conditions, but the difference between DTPA alone and both (DTPA and Met) is smaller.

Key results of investigating oxidative stress (MPL-a combination of metal, peroxide and 3 days of light in addition to thermal stress at 30 ℃) with various formulation combinations demonstrate that there is benefit in formulations with both 50 μm DTPA and 5mM Met. Having only one of these antioxidants is less stable than having both a chelating agent and a sacrificial antioxidant. These results are consistent with the results of study 1.

Results-study 3

In this study, the goal was to understand the behavior of molecules in different packaging components (drug-loaded syringe and patch pump). In addition, there are several formulation composition differences from the initial formulation composition to determine the range over which the benefits of such a formulation can be observed. These include:

Histidine range 15-100 mM. Study 2 showed an increase in HMW at low histidine concentrations of 10 mM.

From a broader enzyme level of 0-10,000U/mL, only up to 5,000U/mL was covered compared to study 2.

A wider protein concentration from 100-200 mg/mL. Study 2 covered up to 150mg/mL.

This study also uses these to confirm the formulation latitude in the case of absence of enzyme, formulation: pH:5.2-6.5His; histidine: 15-100mM; DTPA: 10-200. Mu.M (Alt: 100. Mu.M EDTA); met:1-20mM (Alt: 10mM Tryp); PS80:0.01-0.1% w/v (Alt: PS20 and poloxamer 0.2 mg/mL); sugar: 10-400mM sucrose (Alt: 10% sorbitol and trehalose); protein: 100-200mg/mL; and (3) main packaging: vials, PFS, and patch pumps.

Note that in this study, sorbitol and trehalose were correctly added to the TFF buffer lost in study 2. Since the enzyme has no effect on HMW formation, these results will be used to understand the performance of using these alternative sugars, as will be the case for enzymes.

This resulted in 46 formulations, with all other factors at the target composition, similar to study 2, each single variable study formulation. In this study, vials and patch pumps were studied primarily at 120mg/mL, and PFS was at a target concentration of 150mg/mL Nivo. The target formulation composition was: 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 2,000U/mL rHuPH20, 0.05% w/v PS80, pH 6.0 with an air headspace. Note that in the design for the center point condition and the condition with 50 μm DTPA and no Met, there are multiple independent formulation formulations.

In this study, stability of Nivo was investigated using SEC, and due to the lack of microparticle data from study 3, microparticle analysis was performed at selected time points (MPL 3M, RT 30M, 5c 6M, and 25c 6M).

Size exclusion chromatography

Reproducibility and enzyme influence

The inclusion of multiple and independent formulation formulations in the design for center point conditions and 0Met conditions, as performed for study 2; however, for study 3, the formulation did not include enzymes. For both study 2 and study 3, the final HMWs of both formulations as determined by SEC at stress and time points are tabulated in table 94. This data shows reproducibility between study 2 and study 3 and between independent formulations within the study. The data are always within 0.1% hmw of each other.

Table 94: study 2 and study 3-high molecular weight species-center and 0Met formulation determined by SEC for the last time point in each stress condition studied for the duplicate samples. Center formulation composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0.0Met composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 0.05% w/vPS, pH 6.0.

This study, as well as study 2 (Table 80), showed that with or without enzyme had no effect on Nivo stability at all enzyme levels studied (0-10,000U/mL) (Table 95). Throughout study 3, there was a tested multiplex composition with and without enzyme. These results are very consistent (table 95, table 96, table 97, table 98, table 99, table 100, fig. 25A and fig. 25B) and allow us to confidently translate knowledge (learning) between conditions with and without enzymes.

Table 95: study 3-high molecular weight species determined by SEC for the last time point in each stress condition studied, for different enzyme levels. The preparation comprises the following components: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0.

Main package

This study evaluates three different types of primary packages: a vial, a drug-carrying syringe and a patch pump. The data for the various primary packages for protein concentrations of 120mg/mL are shown in table 96 in the following center point formulation compositions: 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0. There was no difference in HMW formation due to major packaging differences.

Table 96: study 3-high molecular weight species determined by SEC for the final time point in each stress condition studied for the different primary packages at 120mg/mL Nivo. The preparation comprises the following components: 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0

* The data available to the patch pump is limited due to the limited number of patch pumps.

To understand whether there is any susceptibility due to the PFS component, two additional conditions were tested: a standard BD Neopack syringe was filled with 1ppm tungsten (W) and twice the silicone oil found in a standard BD Neopack syringe. The high molecular weight values of the stress conditions at the final time point are tabulated in Table 97, also having 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0 at a concentration of 150 mg/mL. No increase in HMW due to tungsten or silicone oil was detected.

Table 97: study 3-high molecular weight species determined by SEC for the last time point in each stress condition studied for use of PFS component at 150mg/mL Nivo. The preparation comprises the following components: 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0

Protein concentration

The primary packaging and enzyme levels did not affect HMW formation, as discussed above. HMW formation between package and enzyme levels at the center point formulation composition over the protein concentration range from 100 to 200mg/mL is tabulated together in table 98. The center point formulation composition included 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0. Higher protein concentrations showed higher HMW formation, and this was consistent with other studies at higher protein concentrations and data from study 2 (table 88).

Table 98: study 3-high molecular weight species determined by SEC for the last time point in each stress condition studied for various protein concentrations. The preparation comprises the following components: 20mM histidine, 250mM sucrose, 50. Mu. MDTPA, 5mM Met, 0.05% w/v PS80, pH 6.0

Alternative excipients

In study 3, as an alternative sugar to 250mM (8.6%) sucrose, two alternative sugars were studied: 10% sorbitol and 10% trehalose. The results were studied and are tabulated in table 99 using 0.05% w/v PS80, 0.05% w/v PS20 and 0.2mg/mL of the alternative surfactant of poloxamer. These results are tabulated for study 2 versus study 3, where the difference here is that study 2 has 2,000u/mL rHuPH20 and study 3 has no rHuPH20. Other excipients are at the target composition: the target formulation consisted of 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0. In study 2 with the formulation of trehalose and sorbitol for alternative excipients, there was an error during sample preparation, so no data with enzyme was presented.

Table 99: study 2 and study 3-high molecular weight species determined by SEC for the last time point in each stress condition studied for the alternative vehicle. Study 2 had 2,000U/mL rHuPH20 and study 3 had 0U/mL rHuPH20. Center point composition: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 50. Mu.M DTPA, 5mM Met, 0.05% w/v PS80, pH 6.0.

As an alternative excipient PS80 can be replaced by PS20 or poloxamers with little impact on stability. These results were consistent between the two studies.

Having histidine as a buffer is critical to Nivo stability and cannot be replaced with succinate. In all thermal stress conditions studied (5 ℃ 6M, 25 ℃ 6M and 35 ℃ 3M), there was a significant increase in HMW species when succinate buffer was used, and this was still consistent between the two studies.

Sugar data indicate that sucrose has a superior stabilizing effect relative to sorbitol or trehalose.

As an alternative chelator to 50 μm DTPA, 100 μm EDTA was studied as performed in study 2. DTPA is a better chelator, so higher levels of EDTA were studied here. Similarly, 10mM tryptophan was studied as an alternative sacrificial agent to 5mM methionine. Since the addition of chelating agents and sacrificial oxidants may affect protection against oxidation, these chelating agents and sacrificial oxidants are investigated under both thermal and oxidative stress conditions. For study 2 and study 3, the final HMWs determined by SEC using these alternative excipients are tabulated in table 100 for the final time point for this stress condition, with the difference here being that study 2 has 2,000u/mL rHuPH20 and study 3 has no rHuPH20. The formulation consisted of 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0.

The results of the two studies were comparable to each other. The exception is the condition with 0DTPA and 0Met, where there is a higher variability in how fast this autocatalytic HWM increase occurs. The tabulated HMW% value after 3 months under MPL stress clearly shows the unique benefit of DTPA between the two studies and is a better chelator than EDTA. Similarly, the data shows that Met is better than Trp. Control of the% increase in HMW was optimal when formulated with 5mM Met and 50. Mu.M DTPA. These results were consistent between the three studies.

These studies showed a unique beneficial effect of histidine and sucrose in combination with both Met and DTPA on Nivo stability.

Table 100: study 2 and study 3-alternative excipients for DTPA and Met in each of the stress conditions studied, high molecular weight species determined by SEC for the last time point. The composition comprises: 120mg/mL Nivo, 20mM histidine, 250mM sucrose, 0.05% w/v PS80, pH 6.0. Study 2 had 2,000U/mL rHuPH20 and study 3 had 0U/mL rHuPH20.

Excipient ranges

Similar to study 2, various excipient ranges were studied in study 3. These results are consistent and consistent with study 2 results, as demonstrated by the key findings in study 2, where the effect of Met concentration has a beneficial effect with increasing concentration. The results in all three studies are shown in figure 25. Study 1 only assessed 0 and 5mM Met values, and study 2 focused on conditions with enzyme, and study 3 focused on conditions without enzyme. Throughout this example, it has been demonstrated that the enzyme does not affect Nivo stability, and this finding is again demonstrated here.

Regression plots for all 3 studies are shown in figure 26. The three studies overlap each other very well. The linear approximation of the 0 to 5mM methionine action was 0.19% HMW at the following center point composition: 120mg/mL Nivo, 20mM histidine, 50. Mu.M DTPA, 250mM sucrose, 0.05% w/v PS80, pH 6.0.

In study 2 and study 3, the formulation composition ranges that maintain the best stability are as follows: pH:5.2-6.5His; histidine: 15-100mM; DTPA: 10-200. Mu.M (Alt: 100. Mu.M EDTA); met:1-20mM (Alt: 10mM Trp); rHuPH20:0-5,000U/mL; PS80:0.01-0.1% w/v (Alt: PS 20.05% w/v and poloxamer 0.2 mg/mL); sugar: 10-400mM sucrose; protein: 100-175mg/mL; and (3) main packaging: vials, PFS, patch pumps.

The pH range of this formulation is critical. Above pH 6.5, an increase in HMW was observed, and this was consistent in both study 2 and study 3. Formulations closer to pH 6 were suggested based on the data.

Histidine is critical in formulation as a buffer and has a stabilizing effect. The data from study 2 and study 3 are consistent. Succinate as buffer does not show sufficient stability. Lower histidine concentrations lead to higher HMW formation. Near 15mM histidine levels, cliffs with reduced stability are present. In this study, up to 100mM histidine was studied, but higher concentrations were expected to be stable.

The enzyme range between 0 and 10,000U/mL has been studied and does not affect the stability of Nivo. This was consistent between study 2 and study 3.

PS80 levels between 0-0.1% w/v prevent Nivo from HMW formation. The alternative surfactants PS20 (0.05% w/v) and poloxamer (0.2 mg/mL) showed good protection of Nivo against HMW formation.

Nivo with sucrose levels between 10-400mM showed good stability. As an alternative excipient, trehalose and sorbitol show higher HMW formation after storage as compared to sucrose.

No effect on stability due to the primary packaging was observed between the vials tested, PFS and the patch pump.

DTPA with a concentration in the range of 10-200. Mu.M DTPA showed the same stability. Even at a level of 10 μm lower is sufficient to protect Nivo. No concentration dependence was observed between study 2 and study 3.

Increased Met levels showed acceptable stability in the range of 1-20mM Met. The results were consistent between the studies (fig. 26). For the case of Met, a statistical model was constructed. For MPL stress conditions, linear regression was found to be significant for both study 2 and study 3. This shows that there is a clear benefit with Met and that higher concentrations of Met are advantageous for resisting MPL stress.

Microparticles

Study 2 did not include microparticle analysis. In study 3, microparticle analysis was performed at selected time points (MPL 3M, RT 30M, 5 ℃ 6M and 25 ℃ 6M). One sample had an elevated particle count: the sample in which histidine was replaced with succinate as buffer, formulation 25. HMW at 25℃for 6 months showed 6527 particles/mL.gtoreq.10. Mu.m. The remaining samples had less than 130 particles/mL ≡10μm, well below the USP <788> particle specification limit. These data show the problem of converting to succinate as buffer, even if all other formulation components are present.

Conclusion-study 3

In study 3, different packaging components (drug-carrying syringe and patch pump) were studied in addition to the vials and confirmed that they performed well with the formulations.

The aim of the study was also to understand the full range of stability maintenance optimizations. The ranges found (from the combination of study 2 and study 3) are as follows: pH: 5.2-6.5 His; histidine: 15-100mM; DTPA: 10-200. Mu.M (Alt: 100. Mu.M EDTA); met:1-20mM (Alt: 10mM Trp); rHuPH20:0-5,000U/mL; PS80:0.01-0.1% w/v (Alt: PS 20.05% w/v and poloxamer 0.2 mg/mL); sugar: 10-400mM sucrose; protein: 100-175mg/mL; and (3) main packaging: vials, PFS, patch pumps. For samples without enzyme, these ranges are the same.

The center point formulation is: 120mg/mL Nivo (for vials) and 150mg/mL Nivo (for PFS, both containing 20mM histidine, 250mM sucrose, 5mM Met, 50. Mu.M DTPA, 0.05% w/v polysorbate 80, pH 6.0, and 2,000U/mL rHuPH20.

The pH range of this formulation is critical. Above pH 6.5, an increase in HMW was observed. Histidine is critical in formulation as a buffer and has a stabilizing effect. Lower histidine concentrations lead to higher HMW formation. Near 15mM histidine levels, cliffs with reduced stability are present. In this study, up to 100mM histidine was studied, but higher concentrations were expected to be stable. The presence of DTPA was important in maintaining Nivo stability, but no concentration dependence was observed. An increase in Met shows a higher protection for Nivo. There is a clear benefit to having Met and higher concentrations of Met are advantageous for resisting MPL stress.

Succinate as buffer did not show sufficient stability towards Nivo, both forming soluble aggregates (increased HMW as determined by SEC) and insoluble aggregates (particles as determined by MFI). As an alternative stabilizer for sucrose, trehalose and sorbitol showed higher HMW formation after storage. EDTA is not as good as DTPA and Tryp is not as good as Met, even at higher concentrations. These studies showed the unique beneficial effect of histidine and sucrose in combination with Met and DTPA to improve Nivo stability.

Example 6 detection of Nawuzumab-excipient interactions Using Nuclear Magnetic Resonance (NMR) spectroscopy and Molecular Dynamics (MD)

NMR experiments and computational modeling have been performed to study the nivolumab-excipient interactions. Both methods confirm that there should be preferential protein-sugar interactions and show a stronger binding behavior of sucrose relative to mannitol, trehalose, glycine, sorbitol or succinate. The results may indicate the key molecular mechanism of the role of sugar in protein formulations.

Sugar is used to stabilize protein formulations and prevent protein aggregate formation. The goal of this example was to use a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) methods to better understand the mechanism of stabilization, determine why some sugars work better than others, and help guide formulation selection. Protein-ligand interactions and binding were measured using an STD (saturation transfer difference) NMR experiment (see Angulo j. Et al, chem. Eur. J.,16:7803-7812 (2010), which is incorporated herein by reference in its entirety).

NMR data acquisition in ICONNMR was used on a NEO 700Bruker NMR spectrometer and STD NMR experiments were performed using the pulse sequence stddiff at 283K. A recirculation delay of 3-4.5 seconds is used. NMR experiments were performed on samples of nivolumab (21.3 mg/ml concentration) with excipients glycine, mannitol, sucrose, trehalose, sorbitol and succinate to obtain binding efficiency of each saccharide to nivolumab. Experiments were performed at a range of excipient concentrations and the intensities of the integrated peaks in the different spectra were fitted to the Michaelis-Menten equation to assess whether the interaction between excipient and protein was specific. Specific interactions produce saturation effects of signals in different spectra.

Molecular Dynamics (MD) is a computational method that mimics the interaction between molecules at the atomic level. Here, the simulation is an arrangement containing one nano Wu Shankang Fab group, histidine buffer (15 mM equivalent) and excipient molecules (about 190mM equivalent). Each excipient molecule was studied in a separate simulation. Simulation chambers were set up using MOE 2019.0101 software (Molecular Operating Environment 2019.0101;Chemical Computing Group Inc., montreal, canada, 2017) and simulations were run using NAMD 2.12 (Phillips, J.C. et al, J.Comp. Chem,26:1781-1802 (2005)).

MD results were analyzed by first counting how many excipient molecules are within the hydrogen bonding distance (< 2A) from any atom of the Fab group. To distinguish tightly bound interaction poses of excipients from lighter binding interaction poses, a cluster analysis was performed to group similar binding poses of each individual excipient and rank by frequency at those poses in the simulation.

STD NMR results showed that sucrose has the smallest Kd (dissociation constant) value relative to trehalose, glycine and mannitol, sorbitol and succinate, showing the strongest interaction with the nivolumab mAb (fig. 26).

FIG. 27 shows the residues at the Na Wu Shankang Fab group during the final 8ns of MD simulationAverage number of internal sugar molecules. Significant differences were observed between the different excipients, with the sucrose molecules interacting the most and glycine the least. The general trend in the number of interactions is: sucrose>Mannitol = trehalose>Glycine. Sucrose molecules interact more frequently than five times more than glycine and about 50% more than mannitol with a number of sub-multiple interactions. Qualitatively, these results are consistent with NMR results, but because STD-NMR experiments are sensitive to binding strength, not just interactions, the interaction strength is further explored in MD using cluster analysis.

Fig. 28A-28C show the location of unique binding sites identified for each excipient by cluster analysis. Visually, the differences between excipients were evident, with the number of binding sites for mannitol (fig. 28C), trehalose (fig. 28E) and sucrose (fig. 28D) being significantly greater than sorbitol (fig. 28B) and glycine (fig. 28A), matching the trend in fig. 27. Fig. 29A-29B show the number of unique sites identified for each excipient, with fig. 29A being weak-medium binding sites and fig. 29B being strong binding sites. Only sucrose, trehalose and sorbitol have strong binding sites in the simulation, with trehalose and sorbitol having 1 such site and sucrose having 7 such sites. Further analysis of these sites has shown that in these strong binding poses, 3-4 hydrogen bonds are formed between the protein and excipient molecules, verifying that these poses are very advantageous and likely good candidates for exchange sites identified by STD-NMR results. It should be noted that the strongly binding trehalose and sorbitol sites are located in the constant domain of the Fab and close to the hinge region where additional steric interactions may exist that prevent binding in the intact mAb.

NMR results showed that only sucrose interacted/bound strongly with nivolumab compared to glycine, mannitol, sucrose, trehalose, sorbitol and succinate. MD results indicate that all sugars studied interact to some extent, with preference: sucrose > mannitol > trehalose > glycine > sorbitol. MD cluster analysis showed that only sucrose and trehalose had strong binding interactions, which were stabilized by nonspecific and specific interactions with exposed residues on Fab. Observations of only strong binding sites for sucrose correlate NMR observations with MD results, so the differential behavior of sucrose (Kamerzell, t.j. Et al, advanced Drug Delivery Reviews,63:1118-1159 (2011)) makes it a better excipient for stabilizing proteins. The greater overall number of sucrose binding sites may explain why sucrose shows optimal inhibition of aggregation of the tested molecules.

Example 7 analysis of additional hyaluronidase and Nawuzumab

The nivolumab subcutaneous formulation included hyaluronidase (rHuPH 20). In the current study, the compatibility of nivolumab with alternative analogues of rHuPH20 will be evaluated. The alternative enzyme variants were placed in steady state (e.g., at 5 ℃, 25 ℃ or 35 ℃) in the current formulation composition as follows: 120mg/mL of nivolumab in 20mM histidine (pH 6.0), 250mM sucrose, 0.05% polysorbate 80, 5mM methionine, 50. Mu.M pentetic acid and 2000U/mL rHuPH 20.

Various alternative analogs of rHuPH20 in combination with the nivolumab subcutaneous formulation will be evaluated. Examples of alternative analogues to be considered include, but are not limited to, enzymes having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOS: 5-263. For example, an alternative analog of rHuPH20 having the amino acid sequence shown in SEQ ID NO. 92 was placed in steady state in the current formulation composition as follows: 120mg/mL of nivolumab in 20mM histidine (pH 6.0), 250mM sucrose, 0.05% polysorbate 80, 5mM methionine, 50. Mu.M pentetic acid, and rHuPH20 (e.g., 2000U/mL); wherein said rHuPH20 has the amino acid sequence set forth in SEQ ID NO. 92.

The following parameters will be measured for each time point: pH, protein concentration, and SEC. The selected samples will be tested for particulate matter (using HIAC) and enzyme activity. Enough samples will be kept for PS-80 analysis as well as analysis by CD-SDS and iCiEF, and testing will be performed if necessary. For each time point, 6mL will be prepared for analysis.

A total of 22 vials will be filled with 3mL of each sample to cover 11 time points. This will be done in the following way: samples were prepared with 80mL formulation to compensate for losses during filtration using a 0.22 μm PES filter. The filtered drug product was filled into depyrogenated 3cc vials (Schott type 1 glass) and stoppered with 13mm Daikyo stoppers (D-21-7S, fluotec coated serum) treated with high pressure steam. The vial will be screwed on and placed stably in an upright position.

Claims

1. A method of treating a subject in need thereof, the method comprising subcutaneously administering to the subject a dose of a pharmaceutical composition comprising (i) an antibody that specifically binds PD-1 or PD-L1 and inhibits the interaction of PD-1 with PD-L1 (an "anti-PD-1 antibody" or an "anti-PD-L1 antibody", respectively) and (ii) an endoglycosidase hydrolase; wherein the dose comprises one or more subcutaneous unit doses; and wherein the dose comprises at least about 300mg to at least about 2400mg of the anti-PD-1 antibody or the anti-PD-L1 antibody.

2. The method of claim 1, wherein the dose comprises two or more subcutaneous unit doses, and wherein the two or more subcutaneous unit doses are administered in parallel or subsequently.

3. The method of claim 2, wherein the two or more subcutaneous unit doses are administered subsequently, wherein each of the two or more subcutaneous unit doses is administered within an interval of about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about nine hours, about twelve hours, about eighteen hours, or about twenty four hours between the subcutaneous unit doses.

4. The method of any one of claims 1 to 3, wherein the dose is administered about every, two, three, four, six, eight weeks.

5. The method of any one of claims 1-4, wherein the antibody comprises an anti-PD-1 antibody.

6. The method of any one of claims 1 to 5, wherein the dosage of the antibody is about 250mg to about 600mg administered about weekly.

7. The method of any one of claims 1-6, wherein the antibody is administered at a dose of about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, or about 600mg per week.

8. The method of any one of claims 1 to 7, wherein the dosage of the antibody is about 300mg administered about weekly.

9. The method of claim 8, wherein the dosage of antibody comprises a single subcutaneous unit dose of about 300mg.

10. The method of claim 8 or 9, wherein the dosage of antibody comprises a single subcutaneous unit dose of about 300mg in a total administration volume of about 2 mL.

11. The method of claim 10, wherein the dosage of the antibody comprises (i) two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 150mg of the antibody; or (ii) three subcutaneous unit doses, wherein each of the three subcutaneous unit doses comprises about 100mg of the antibody.

12. The method of claim 11, wherein (i) the two subcutaneous unit doses are administered to the same body location of the subject, or (ii) at least two of the three subcutaneous unit doses are administered to the same body location of the subject.

13. The method of any one of claims 1 to 5, wherein the antibody is administered at a dose of about 300mg to about 900mg every two weeks.

14. The method of claim 13, wherein the antibody is administered at a dose of about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 510mg, about 520mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg, about 600mg, about 610mg, about 620mg, about 630mg, about 640mg, about 650mg, about 660mg, about 670mg, about 680mg, about 690mg, about 700mg, about 710mg, about 720mg, about 730mg, about 740mg, about 750mg, about 760mg, about 770mg, about 780mg, about 790mg, about 800mg, about 810mg, about 820mg, about 830mg, about 840mg, about 850mg, about 860mg, about 870mg, about 880mg, about 890mg, or about 900mg every two weeks.

15. The method of claim 13 or 14, wherein the antibody is administered at a dose of about 600mg every two weeks.

16. The method of claim 15, wherein the dosage of antibody comprises a single subcutaneous unit dosage.

17. The method of claim 15, wherein the dosage of antibody comprises two, three, or at least four subcutaneous unit doses.

18. The method of claim 15 or 17, wherein the dose of the antibody comprises two subcutaneous unit doses, wherein each of the two subcutaneous unit doses comprises about 300mg of the antibody.

19. The method of claim 18, wherein at least one of the two subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL.

20. The method of any one of claims 17 to 19, wherein at least two of the subcutaneous unit doses are administered to the same body location of the subject.

21. The method of any one of claims 1 to 5, wherein the antibody is administered at a dose of about 900mg to about 1500mg every four weeks.

22. The method of claim 21, wherein the antibody is administered at a dose of about 900, about 950, about 1000, about 1010, about 1020, about 1030, about 1040, about 1050, about 1060, about 1070, about 1080, about 1090, about 1100, about 1110, about 1120, about 1130, about 1140, about 1150, about 1160, about 1170, about 1180, about 1190, about 1200, about 1210, about 1220, about 1230, about 1240, about 1250, about 1260, about 1270, about 1280, about 1290, about 1300, about 1310, about 1320, about 1330, about 1340, about 1350, about 1360, about 1370, about 1380, about 1390, about 1400, about 1410, about 1430, about 1440, about 1450, about 40, about 1460, about 1490, or about 1500.

23. The method of claim 21 or 22, wherein the antibody is administered at a dose of about 1200mg every four weeks.

24. The method of any one of claims 21 to 23, wherein the dose of antibody comprises a single subcutaneous unit dose.

25. The method of any one of claims 21 to 23, wherein the dose of antibody comprises two, three, four, six, or at least eight subcutaneous unit doses.

26. The method of any one of claims 21 to 23 or 25, wherein the dose of antibody comprises four subcutaneous unit doses, wherein each of the four subcutaneous unit doses comprises about 300mg of the antibody.

27. The method of claim 26, wherein at least one of the four subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of 2 mL.

28. The method of any one of claims 25 to 27, wherein at least two of the subcutaneous unit doses are administered to the same body location of the subject.

29. The method of any one of claims 25 to 28, wherein the two, three, four, six, or at least eight subcutaneous unit doses are administered on the same day.

30. The method of any one of claims 1-4, wherein the antibody comprises an anti-PD-L1 antibody.

31. The method of claim 30, wherein the dosage of the antibody is about 900mg to about 1800mg of the antibody administered every two weeks.

32. The method of claim 30 or 31, wherein the antibody is administered at a dose of about 900, about 950, about 1000, about 1010mg, about 1020mg, about 1030mg, about 1040mg, about 1050mg, about 1060mg, about 1070mg, about 1080mg, about 1090mg, about 1100mg, about 1110mg, about 1120mg, about 1130mg, about 1140mg, about 1150mg, about 1160mg, about 1170mg, about 1180mg, about 1190mg, about 1200mg, about 1210mg, about 1220mg, about 1230mg, about 1240mg, about 1250mg, about 1260mg, about 1270mg, about 1280mg, about 1290mg, about 1300mg, about 1310mg, about 1320mg, about 1330mg, about 1340mg, about 1350mg, about 1360mg, about 1370mg, about 1380mg, about 1390mg, about 1400mg, about 1430mg, about 1440mg, about 1450mg, about 1460mg, about 148 mg, about 1500mg.

33. The method of any one of claims 30 to 32, wherein the dosage of the antibody is about 1200mg every two weeks.

34. The method of any one of claims 30-33, wherein the dose comprises a single subcutaneous unit dose of greater than about 1200mg in a total administration volume of about 5 mL.

35. The method of any one of claims 30 to 33, wherein the dose comprises two, three, four, six, or at least eight subcutaneous unit doses.

36. The method of any one of claims 30 to 33 and 25, wherein the dose of the antibody comprises four subcutaneous unit doses, wherein each of the four subcutaneous unit doses comprises about 300mg of the antibody.

37. The method of claim 36, wherein at least one of the four subcutaneous unit doses comprises greater than about 300mg of the antibody in a total volume of about 2 mL.

38. The method of any one of claims 35 to 37, wherein at least two of the subcutaneous unit doses are administered to the same body location of the subject.

39. The method of any one of claims 35 to 38, wherein the two, three, four, six, or at least eight subcutaneous unit doses are administered on the same day.

40. The method of any one of claims 1-39, wherein the anti-PD-1 antibody or anti-PD-L1 antibody and the endoglycosidase hydrolase are administered together.

41. The method of any one of claims 1-39, wherein the anti-PD-1 antibody or anti-PD-L1 antibody and the endoglycosidase hydrolase are administered in parallel.

42. The method of any one of claims 1-39, wherein the anti-PD-1 antibody or anti-PD-L1 antibody and the endoglycosidase hydrolase are administered sequentially.

43. The method of any one of claims 1 to 42, wherein the pharmaceutical composition is administered at a dose of at least about 1,000 units to at least about 30,000 units of the endoglycosidase hydrolase.

44. The method of any one of claims 1-43, wherein the pharmaceutical composition is a dosage of the enzyme that is administered in at least about 1000 units, at least about 1500 units, at least about 2000 units, at least about 2500 units, at least about 3000 units, at least about 3500 units, at least about 4000 units, at least about 4500 units, at least about 5000 units, at least about 6000 units, at least about 7000 units, at least about 8000 units, at least about 9000 units, at least about 10,000 units, at least about 11,000 units, at least about 12,000 units, at least about 13,000 units, at least about 14,000 units, at least about 15,000 units, at least about 16,000 units, at least about 17,000 units, at least about 18,000 units, at least about 19,000 units, at least about 20,000 units, at least about 21,000 units, at least about 22,000 units, at least about 23,000 units, at least about 24,000 units, at least about 25,000 units, at least about 26,000 units, at least about 27,000 units, at least about 28,000 units, at least about 29,000 units, or at least about 30,000 units.

45. The method of any one of claims 1 to 44, wherein the pharmaceutical composition is administered at a dose of about 4000 units of the endoglycosidase hydrolase.

46. The method of any one of claims 1-12 and 40-45, wherein the pharmaceutical composition is administered at a dose of: (i) About 300mg of the antibody and (ii) about 4000 units of the endoglycosidase hydrolase, about once a week.

47. The method of any one of claims 1-20 and 40-45, wherein the pharmaceutical composition is administered at the following doses: (i) About 600mg of the antibody and (ii) about 8000 units of the endoglycosidase hydrolase, about once every two weeks.

48. The method of any one of claims 1 to 29 and 40 to 45, wherein the pharmaceutical composition is administered at the following doses: (i) About 1200mg of the antibody and (ii) about 20,000 units of the endoglycosidase hydrolase, about once every four weeks.

49. The method of any one of claims 1 and 30-45, wherein the pharmaceutical composition is administered at a dose of: (i) About 1200mg of the antibody and (ii) about 8000 units of the endoglycosidase hydrolase, about once every two weeks.

50. The method of any one of claims 1 and 30-45, wherein the pharmaceutical composition is administered at a dose of: (i) About 1800mg of said antibody and (ii) about 12,000 units of said endoglycosidase hydrolase, about once every three weeks.

51. The method of any one of claims 1 to 50, wherein the endoglycosidase hydrolase cleaves hyaluronic acid at a hexosamine β (1-4) or (1-3) junction.

52. The method of any one of claims 1 to 51, wherein the endoglycosidase hydrolase comprises the catalytic domain of hyaluronidase PH-20 (HuPH 20), HYAL1, HYAL2, HYAL3, HYAL4, or HYALPS 1.

53. The method of any one of claims 1 to 52, wherein the endoglycosidase hydrolase comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to amino acids 36-490 of SEQ ID No. 1.

54. The method of any one of claims 1 to 53, wherein the endoglycosidase hydrolase comprises a hyaluronidase.

55. The method of any one of claims 1 to 54, wherein the endoglycosidase hydrolase comprises a hyaluronidase selected from the group consisting of HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, any variant thereof, and any isoform thereof.

56. The method of any one of claims 1-55, wherein the endoglycosidase hydrolase comprises rHuPH20 or a fragment thereof.

57. The method of any one of claims 1 to 56, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

58. The method of any one of claims 1 to 57, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the alpha helical region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

59. The method of any one of claims 1 to 58, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the linker region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

60. The method of any one of claims 1 to 59, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase in which one or more N-terminal and/or C-terminal amino acids are deleted relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

61. The method of any one of claims 1-60, wherein the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises:

i. one or more amino acid substitutions in the alpha helical region, the linker region, or both the alpha helical region and the linker region relative to wild-type rHuPH 20;

deletion of one or more N-terminal amino acids, one or more C-terminal amino acids, or one or more N-terminal amino acids and one or more C-terminal amino acids relative to wild-type rHuPH 20; or alternatively

Both (i) and (ii).

62. The method of any one of claims 1-29 and 40-61, wherein the anti-PD-1 antibody comprises an antibody selected from the group consisting of: nivolumab, pembrolizumab, PDR001, MEDI-0680, cimetidine Li Shan, terlipressin Li Shan, tirelimumab, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, BCD-100, IBI308, and any combination thereof.

63. The method of claim 62, wherein the anti-PD-1 antibody cross-competes with nivolumab for binding to human PD-1.

64. The method of claim 62, wherein the anti-PD-1 antibody comprises nivolumab.

65. The method of claim 62, wherein the anti-PD-1 antibody comprises pembrolizumab.

66. The method of any one of claims 1 and 30-61, wherein the anti-PD-L1 antibody comprises an antibody selected from the group consisting of: BMS-936559, ab, duvaluzumab, ab, STI-1014, CX-072, KN035, LY3300054, BGB-A333, CK-301, and any combination thereof.

67. The method of any one of claims 1-66, wherein the subject has cancer.

68. The method of claim 67, wherein the step of, wherein the cancer is selected from squamous cell carcinoma (squamous cell carcinoma), small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (renal cancer), clear cell carcinoma, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer (endometrial cancer), renal cancer (kidney cancer), renal Cell Carcinoma (RCC), prostate cancer, hormone refractory prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer (cancer), gastric cancer (cancer), bladder cancer, hepatoma, breast cancer, colon cancer, head and neck cancer, gastric cancer (cancer), germ cell tumor, pediatric sarcoma natural killer cells of the sinuses, melanoma, bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, fallopian tube cancer, endometrial cancer (carcinoma of the endometrium), cervical cancer (carcinoma of the cervix), vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, rectal cancer, solid tumors in children, ureter cancer, renal pelvis cancer, central Nervous System (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma (squamous cell cancer), environmentally induced cancers including induced cancer asbestos, asbestos, virus-associated cancer or virus-derived cancer (e.g., human papillomavirus (HPV-associated or derived tumor)), and any combination thereof.

69. The method of any one of claims 1-68, wherein the pharmaceutical composition is administered using an auto-injector.

70. The method of any one of claims 1-68, wherein the pharmaceutical composition is administered using a wearable pump.

71. The method of any one of claims 1-70, wherein the pharmaceutical composition is administered to the subject by subcutaneous infusion for less than about 10 minutes.

72. The method of any one of claims 1-71, wherein the pharmaceutical composition is administered to the subject by subcutaneous infusion for less than about 5 minutes.

73. The method of any one of claims 1-72, wherein the pharmaceutical composition further comprises at least two antioxidants.

74. The method of claim 73, wherein the at least two antioxidants are selected from methionine, tryptophan, histidine, cysteine, ascorbic acid, glycine, DTPA and EDTA.

75. The method of claim 73 or 74, wherein the at least two antioxidants comprise (i) methionine and EDTA, or (ii) methionine and DTPA.

76. The method of any one of claims 73-75, wherein the at least two antioxidants comprise at least about 1 to about 20mM methionine.

77. The method of any one of claims 73-76, wherein the at least two antioxidants comprise at least about 1mM, at least about 1.5mM, at least about 2mM, at least about 2.5mM, at least about 3mM, at least about 3.5mM, at least about 4mM, at least about 4.5mM, at least about 5mM, at least about 5.5mM, at least about 6mM, at least about 6.5mM, at least about 7mM, at least about 7.5mM, at least about 8mM, at least about 8.5mM, at least about 9mM, at least about 9.5mM, at least about 10mM, at least about 11mM, at least about 12mM, at least about 13mM, at least about 14mM, at least about 15mM, at least about 16mM, at least about 17mM, at least about 18mM, at least about 19mM, or at least about 20mM methionine.

78. The method of any one of claims 73-77, wherein the at least two antioxidants comprise about 5mM methionine.

79. The method of any one of claims 73-78, wherein the at least two antioxidants comprise at least about 10 μΜ to about 200 μΜ DTPA.

80. The method of any one of claims 73-79, wherein the at least two antioxidants comprise at least about 10 μΜ, at least about 15 μΜ, at least about 20 μΜ, at least about 25 μΜ, at least about 30 μΜ, at least about 35 μΜ, at least about 40 μΜ, at least about 45 μΜ, at least about 50 μΜ, at least about 55 μΜ, at least about 60 μΜ, at least about 65 μΜ, at least about 70 μΜ, at least about 75 μΜ, at least about 80 μΜ, at least about 85 μΜ, at least about 90 μΜ, at least about 95 μΜ, at least about 100 μΜ, at least about 110 μΜ, at least about 120 μΜ, at least about 130 μΜ, at least about 140 μΜ, at least about 150 μΜ, at least about 160 μΜ, at least about 170 μΜ, at least about 180 μΜ, at least about 190 μΜ, or at least about 200 μΜ DTPA.

81. The method of any one of claims 73-80, wherein the at least two antioxidants comprise about 50 μΜ DTPA.

82. The method of any one of claims 1-81, wherein the pharmaceutical composition further comprises a tonicity modifier and/or stabilizer.

83. The method of claim 66, wherein the tonicity adjusting agent and/or stabilizing agent comprises a sugar, amino acid, polyol, salt or combination thereof.

84. The method of claim 82 or 83, wherein the tonicity modifier and/or stabilizer is selected from the group consisting of sucrose, sorbitol, trehalose, mannitol, glycerol, glycine, leucine, isoleucine, sodium chloride, proline, arginine, histidine, and any combination thereof.

85. The method of any one of claims 82-84, wherein the tonicity modifying agent comprises sucrose.

86. The method of any one of claims 1-85, wherein the pharmaceutical composition comprises at least about 10mM to at least about 500mM sucrose.

87. The method of any one of claims 1-86, wherein the pharmaceutical composition comprises at least about 10mM, at least about 20mM, at least about 30mM, at least about 40mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, at least about 100mM, at least about 110mM, at least about 120mM, at least about 130mM, at least about 140mM, at least about 150mM, at least about 160mM, at least about 170mM, at least about 180mM, at least about 190mM, at least about 200mM, at least about 210mM, at least about 220mM, at least about 230mM, at least about 240mM, at least about 250mM, at least about 260mM, at least about 270mM, at least about 280mM, at least about 290mM, at least about 300mM, at least about 310mM, at least about 320mM, at least about 330mM, at least about 340mM, at least about 350mM, at least about 360mM, at least about 370mM, at least about 380mM, at least about 390mM, at least about 400mM, at least about 410mM, at least about 420mM, at least about 460mM, at least about 470mM, at least about 440mM, at least about 500mM, or at least about 500 mM.

88. The method of any one of claims 1-87, wherein the pharmaceutical composition comprises about 250mM sucrose.

89. The method of any one of claims 1-88, wherein the pharmaceutical composition further comprises a buffer.

90. The method of claim 89, wherein said buffer is selected from the group consisting of histidine, succinate, tromethamine, sodium phosphate, sodium acetate and sodium citrate.

91. The method of claim 89 or 90, wherein the buffer comprises histidine.

92. The method of any one of claims 1-91, wherein the pharmaceutical composition comprises at least about 5mM to at least about 100mM histidine.

93. The method of any one of claims 1-92, wherein the pharmaceutical composition comprises at least about 5mM, at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, or at least about 100mM histidine.

94. The method of any one of claims 1-93, wherein the pharmaceutical composition comprises about 20mM histidine.

95. The method of any one of claims 1-94, wherein the pharmaceutical composition further comprises a surfactant.

96. The method of claim 95, wherein the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188.

97. The method of claim 95 or 96, wherein said surfactant comprises polysorbate 80.

98. The method of any one of claims 1 to 97, wherein the pharmaceutical composition comprises at least about 0.01% w/v to at least about 0.1% w/v polysorbate 80.

99. The method of any one of claims 1-98, wherein the pharmaceutical composition comprises at least about 0.01% w/v, at least about 0.02% w/v, at least about 0.03% w/v, at least about 0.04% w/v, at least about 0.05% w/v, at least about 0.06% w/v, at least about 0.07% w/v, at least about 0.08% w/v, at least about 0.09% w/v, or at least about 0.1% w/v polysorbate 80.

100. The method of any one of claims 1-99, wherein the pharmaceutical composition comprises about 0.05% w/v polysorbate 80.

101. The method of any one of claims 1 to 29 and 40 to 100, wherein the pharmaceutical composition comprises:

(a) About 120mg/mL of the anti-PD-1 antibody;

(b) About 20mM L-histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 0.0182mg/mL rHuPH20.

102. The method of any one of claims 1 to 29 and 40 to 100, wherein the pharmaceutical composition comprises:

(a) About 120mg/mL of the anti-PD-1 antibody;

(b) About 20mM L-histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 2000U/mL rHuPH20.

103. The method of any one of claims 1 to 29 and 40 to 100, wherein the pharmaceutical composition comprises:

(a) About 150mg/mL of the anti-PD-1 antibody;

(b) About 20mM L-histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 0.0182mg/mL rHuPH20.

104. The method of any one of claims 1 to 29 and 40 to 100, wherein the pharmaceutical composition comprises:

(a) About 150mg/mL of the anti-PD-1 antibody;

(b) About 20mM L-histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 2000U/mL rHuPH20.

105. The method of any one of claims 1-104, wherein the pharmaceutical composition comprises a pH of about 5.2 to about 6.8.

106. The method of any one of claims 1-105, wherein the pharmaceutical composition comprises a pH of 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 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, or about 6.8.

107. The method of any one of claims 1-106, wherein the pharmaceutical composition comprises a pH of about 6.0.

108. A pharmaceutical composition for use in the method of any one of claims 1 to 107.

109. A pharmaceutical composition comprising (i) an antibody that specifically binds PD-1 ("anti-PD-1 antibody"), (ii) an endoglycosidase hydrolase, and (iii) at least two antioxidants.

110. The pharmaceutical composition of claim 109, comprising at least about 50U/mL to at least about 5000U/mL of the endoglycosidase hydrolase.

111. The pharmaceutical composition of claim 109 or 110, comprising the glycosidase enzyme of at least about 50U/mL, at least about 100U/mL, at least about 150U/mL, at least about 200U/mL, at least about 250U/mL, at least about 300U/mL, at least about 350U/mL, at least about 400U/mL, at least about 450U/mL, at least about 500U/mL, at least about 750U/mL, at least about 1000U/mL, at least about 1500U/mL, at least about 2000U/mL, at least about 2500U/mL, at least about 3000U/mL, at least about 3500U/mL, at least about 4000U/mL, at least about 4500U/mL, at least about 5000U/mL, at least about 5500U/mL, at least about 6000U/mL, at least about 6500U/mL, at least about 7000U/mL, at least about 7500U/mL, at least about 8000U/mL, at least about 8500U/mL, at least about 9000U/mL, at least about 9500U/mL, at least about 10,000U/mL.

112. The pharmaceutical composition of any one of claims 109-111, comprising at least about 500U/mL of the endoglycosidase hydrolase.

113. The pharmaceutical composition of any one of claims 109-111, comprising at least about 1000U/mL of the endoglycosidase hydrolase.

114. The pharmaceutical composition of any one of claims 109-111, comprising at least about 2000U/mL of the endoglycosidase hydrolase.

115. The pharmaceutical composition of any one of claims 109-114, wherein the endoglycosidase hydrolase cleaves hyaluronic acid at a hexosamine β (1-4) or (1-3) junction.

116. The pharmaceutical composition of any one of claims 109-115, wherein the endoglycosidase hydrolase comprises the catalytic domain of hyaluronidase PH-20 (HuPH 20), HYAL1, HYAL2, HYAL3, HYAL4, or HYALPS 1.

117. The pharmaceutical composition of any one of claims 109-116, wherein the endoglycosidase hydrolase comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity to amino acids 36-490 of SEQ ID NO: 1.

118. The pharmaceutical composition of any one of claims 109-117, wherein the endoglycosidase hydrolase comprises a hyaluronidase.

119. The pharmaceutical composition of any one of claims 109-118, wherein the endoglycosidase hydrolase comprises a hyaluronidase selected from the group consisting of HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, any variant thereof, and any isoform thereof.

120. The pharmaceutical composition of any one of claims 109-119, wherein the endoglycosidase hydrolase comprises rHuPH20 or a fragment thereof.

121. The pharmaceutical composition of any one of claims 109-120, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

122. The pharmaceutical composition of any one of claims 109-121, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in the alpha helical region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

123. The pharmaceutical composition of any one of claims 109-122, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase comprising one or more amino acid substitutions in a linker region relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

124. The pharmaceutical composition of any one of claims 109-123, wherein the endoglycosidase hydrolase comprises a modified hyaluronidase in which one or more N-terminal and/or C-terminal amino acids are deleted relative to a wild-type hyaluronidase selected from HuPH20, HYAL1, HYAL2, HYAL3, HYAL4, HYALPS1, or fragments thereof.

125. The pharmaceutical composition of any one of claims 109-124, wherein the endoglycosidase hydrolase comprises a modified rHuPH20, wherein the modified rHuPH20 comprises:

Both (i) and (ii).

126. The pharmaceutical composition of any one of claims 109-125, wherein the at least two antioxidants are selected from methionine, tryptophan, histidine, cysteine, ascorbic acid, glycine, DTPA, and EDTA.

127. The pharmaceutical composition of claim 109 or 126, wherein the at least two antioxidants comprise (i) methionine and EDTA, or (ii) methionine and DTPA.

128. The pharmaceutical composition of any one of claims 109-127, wherein the at least two antioxidants comprise at least about 1 to about 20mM methionine.

129. The pharmaceutical composition of any one of claims 109-128, wherein the at least two antioxidants comprise at least about 1mM, at least about 1.5mM, at least about 2mM, at least about 2.5mM, at least about 3mM, at least about 3.5mM, at least about 4mM, at least about 4.5mM, at least about 5mM, at least about 5.5mM, at least about 6mM, at least about 6.5mM, at least about 7mM, at least about 7.5mM, at least about 8mM, at least about 8.5mM, at least about 9mM, at least about 9.5mM, at least about 10mM, at least about 11mM, at least about 12mM, at least about 13mM, at least about 14mM, at least about 15mM, at least about 16mM, at least about 17mM, at least about 18mM, at least about 19mM, or at least about 20mM methionine.

130. The pharmaceutical composition of any one of claims 109-129, wherein the at least two antioxidants comprise about 5mM methionine.

131. The pharmaceutical composition of any one of claims 109-130, wherein the at least two antioxidants comprise at least about 10 μΜ to about 200 μΜ DTPA.

132. The pharmaceutical composition of any one of claims 109-131, wherein the at least two antioxidants comprise at least about 10 μΜ, at least about 15 μΜ, at least about 20 μΜ, at least about 25 μΜ, at least about 30 μΜ, at least about 35 μΜ, at least about 40 μΜ, at least about 45 μΜ, at least about 50 μΜ, at least about 55 μΜ, at least about 60 μΜ, at least about 65 μΜ, at least about 70 μΜ, at least about 75 μΜ, at least about 80 μΜ, at least about 85 μΜ, at least about 90 μΜ, at least about 95 μΜ, at least about 100 μΜ, at least about 110 μΜ, at least about 120 μΜ, at least about 130 μΜ, at least about 140 μΜ, at least about 150 μΜ, at least about 160 μΜ, at least about 170 μΜ, at least about 180 μΜ, at least about 190 μΜ, or at least about 200 μΜ DTPA.

133. The pharmaceutical composition of any one of claims 109-132, wherein the at least two antioxidants comprise about 50 μΜ DTPA.

134. The pharmaceutical composition of any one of claims 109-133, comprising at least about 20mg/mL to at least about 200mg/mL of the anti-PD-1 antibody.

135. The pharmaceutical composition of any one of claims 109-134, comprising at least about 135mg/mL to at least about 180mg/mL of the anti-PD-1 antibody.

136. The pharmaceutical composition of any one of claims 109-135, comprising at least about 108mg/mL to at least about 132mg/mL of the anti-PD-1 antibody.

137. The pharmaceutical composition of any one of claims 109-134, comprising at least about 20mg/mL, at least about 30mg/mL, at least about 40mg/mL, at least about 50mg/mL, at least about 60mg/mL, at least about 70mg/mL, at least about 80mg/mL, at least about 90mg/mL, at least about 100mg/mL, at least about 110mg/mL, at least about 120mg/mL, at least about 130mg/mL, at least about 140mg/mL, at least about 150mg/mL, at least about 160mg/mL, at least about 170mg/mL, at least about 180mg/mL, at least about 190mg/mL, or at least about 200mg/mL of the anti-PD-1 antibody.

138. The pharmaceutical composition of any one of claims 109-137, comprising about 120mg/mL of the anti-PD-1 antibody.

139. The pharmaceutical composition of any one of claims 109-138, comprising about 150mg/mL of the anti-PD-1 antibody.

140. The pharmaceutical composition of any one of claims 109-139, further comprising a tonicity modifier and/or stabilizer.

141. The pharmaceutical composition of claim 140, wherein the tonicity modifier and/or stabilizer includes a sugar, amino acid, polyol, salt or combination thereof.

142. The pharmaceutical composition of claim 140 or 141, wherein the tonicity modifier and/or stabilizer is selected from the group consisting of sucrose, sorbitol, trehalose, mannitol, glycerol, glycine, leucine, isoleucine, sodium chloride, proline, arginine, histidine and any combination thereof.

143. The pharmaceutical composition of any one of claims 140-142, wherein the tonicity modifying agent comprises sucrose.

144. The pharmaceutical composition of any one of claims 109-143, comprising at least about 10mM to at least about 500mM sucrose.

145. The pharmaceutical composition of any one of claims 109-144, comprising at least about 10mM, at least about 20mM, at least about 30mM, at least about 40mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, at least about 100mM, at least about 110mM, at least about 120mM, at least about 130mM, at least about 140mM, at least about 150mM, at least about 160mM, at least about 170mM, at least about 180mM, at least about 190mM, at least about 200mM, at least about 210mM, at least about 220mM, at least about 230mM, at least about 240mM, at least about 250mM, at least about 260mM, at least about 270mM, at least about 280mM, at least about 290mM, at least about 300mM, at least about 310mM, at least about 320mM, at least about 330mM, at least about 340mM, at least about 350mM, at least about 360mM, at least about 370mM, at least about 380mM, at least about 390mM, at least about 400mM, at least about 410mM, at least about 420mM, at least about 470mM, at least about 440mM, at least about 500mM, or at least about 500 mM.

146. The pharmaceutical composition of any one of claims 109-145, comprising about 250mM sucrose.

147. The pharmaceutical composition of any one of claims 109-146, further comprising a buffer.

148. The pharmaceutical composition of claim 147, wherein the buffer is selected from histidine, succinate, tromethamine, sodium phosphate, sodium acetate and sodium citrate.

149. The pharmaceutical composition of claim 147 or 148, wherein the buffer comprises histidine.

150. The pharmaceutical composition of any one of claims 109-149, comprising at least about 5mM to at least about 100mM histidine.

151. The pharmaceutical composition of any one of claims 109-150, comprising at least about 5mM, at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 60mM, at least about 70mM, at least about 80mM, at least about 90mM, or at least about 100mM histidine.

152. The pharmaceutical composition of any one of claims 109-151, comprising about 20mM histidine.

153. The pharmaceutical composition of any one of claims 109-152, further comprising a surfactant.

154. The pharmaceutical composition of claim 153, wherein the surfactant is selected from polysorbate 20, polysorbate 80, and poloxamer 188.

155. The pharmaceutical composition of claim 153 or 154, wherein the surfactant comprises polysorbate 80.

156. The pharmaceutical composition of any one of claims 109-155, comprising at least about 0.01% w/v to at least about 0.1% w/v polysorbate 80.

157. The pharmaceutical composition of any one of claims 109-156, comprising at least about 0.01% w/v, at least about 0.02% w/v, at least about 0.03% w/v, at least about 0.04% w/v, at least about 0.05% w/v, at least about 0.06% w/v, at least about 0.07% w/v, at least about 0.08% w/v, at least about 0.09% w/v, or at least about 0.1% w/v polysorbate 80.

158. The pharmaceutical composition of any one of claims 109-157, comprising about 0.05% w/v polysorbate 80.

159. The pharmaceutical composition of any one of claims 109-158, comprising:

(a) About 150mg/mL of the anti-PD-1 antibody;

(b) About 20mM histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 2000U/mL rHuPH20.

160. The pharmaceutical composition of any one of claims 109-159, comprising:

(a) About 120mg/mL of the anti-PD-1 antibody;

(b) About 20mM histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 2000U/mL rHuPH20.

161. The pharmaceutical composition of any one of claims 109-160, comprising a pH of about 5.2 to about 6.8.

162. The pharmaceutical composition of any one of claims 109-161, comprising a pH of 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 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, or about 6.8.

163. The pharmaceutical composition of any one of claims 109-162, comprising a pH of about 6.0.

164. The pharmaceutical composition of any one of claims 1-163, further comprising a second therapeutic agent.

165. The pharmaceutical composition of claim 164, wherein the second therapeutic agent is an antibody.

166. The pharmaceutical composition of claim 165, wherein the second therapeutic agent is a checkpoint inhibitor.

167. The pharmaceutical composition of claim 166, wherein the checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM 3 antibody, an anti-TIGIT antibody, an anti-TIM 3 antibody, an anti-NKG 2a antibody, an anti-OX 40 antibody, an anti-ICOS antibody, an anti-MICA antibody, an anti-CD 137 antibody, an anti-KIR antibody, an anti-tgfβ antibody, an anti-IL-10 antibody, an anti-IL-8 antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-CXCR 4 antibody, an anti-mesothelin antibody, an anti-CD 27 antibody, an anti-GITR, or any combination thereof.

168. A unit dose comprising the pharmaceutical composition of any one of claims 107-167.

169. A unit dose, the unit dose comprising:

(a) About 120mg/mL of the anti-PD-1 antibody;

(b) About 20mM L-histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 2000U/mL rHuPH20.

170. A unit dose, the unit dose comprising:

(a) About 150mg/mL of the anti-PD-1 antibody;

(b) About 20mM L-histidine;

(c) About 250mM sucrose;

(d) About 0.05% w/v polysorbate 80;

(e) About 50 μm pentetic acid;

(f) About 5mM methionine; and

(g) About 2000U/mL rHuPH20.

171. A vial comprising the pharmaceutical composition of any one of claims 105-167 or the unit dose of any one of claims 168-170.

172. The vial of claim 171, the vial being an auto-injector.

173. An auto-injector comprising the pharmaceutical composition of any one of claims 105-167 or the unit dose of any one of claims 168-170.

174. The vial of claim 171, the vial being a wearable pump.

175. A wearable pump comprising the pharmaceutical composition of any one of claims 105-167 or the unit dose of any one of claims 168-170.