JP2024513123A - Use of anti-TGFβ antibodies and other therapeutic agents to treat proliferative diseases - Google Patents

Abstract

Therapy with TGFβ inhibitors, PD-1 inhibitors, and/or chemotherapeutic agents is disclosed. These drugs at specific doses (including fixed doses) and dosing schedules can be used to treat or prevent proliferative diseases such as solid tumors, including pancreatic or colorectal cancer. Further combinations and uses thereof are also disclosed.

Description

REFERENCES BY INCORPORATION All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference herein. It is hereby incorporated by reference in its entirety for all purposes to the same extent as if indicated. In the event of a conflict between the teachings of one or more of the references incorporated herein and the teachings of this disclosure, the teachings of this specification shall control.

SEQUENCE LISTING This application contains a Sequence Listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. This ASCII copy made on December 17, 2021 is PAT059084_SL. It is named txt and has a size of 350,107 bytes.

The transforming growth factor beta (TGFβ) protein family consists of three different isoforms found in mammals: TGFβ1, TGFβ2, and TGFβ3. TGFβ proteins activate and regulate multiple gene responses that influence disease states, including cell proliferative, inflammatory, and cardiovascular pathologies. TGFβ is a multifunctional cytokine, originally named for its ability to transform normal fibroblasts into cells capable of anchorage-independent growth. TGFβ molecules are primarily produced by hematopoietic and tumor cells, and they control, i.e. stimulate or inhibit, the growth and differentiation of cells from both normal and neoplastic tissues (Sporn et al. ., Science, 233:532 (1986)) and can stimulate the formation and expansion of various stromal cells.

TGFβ is known to be involved in many proliferative and non-proliferative cellular processes, such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses. be. For example, Pircher et al, Biochem. Biophys. Res. Commun. , 136:30-37 (1986); Wakefield et al. , Growth Factors, 1:203-218 (1989); Roberts and Sporn, pp 419-472 in Handbook of Experimental Pharmacology eds M. B. Sporn&A. B. Roberts (Springer, Heidelberg, 1990); Massague et al. , Annual Rev. Cell Biol. , 6:597-646 (1990); Singer and Clark, New Eng. J. Med. , 341:738-745 (1999). TGFβ is also used for the treatment and prevention of intestinal mucosal diseases (International Publication No. 2001/24813 pamphlet). TGFβ also inhibits cytotoxic T lymphocytes (CTL) (Ranges et al., J. Exp. Med., 166:991, 1987), Espevik et al. , J. Immunol. , 140:2312, 1988), suppression of B cell lymphopoiesis and κ light chain expression (Lee et al., J. Exp. Med., 166:1290, 1987), negative regulation of hematopoiesis (Sing et al. , Blood, 72:1504, 1988), downregulation of HLA-DR expression on tumor cells (Czarniecki et al., J. Immunol., 140:4217, 1988), and antigen-induced activation in response to B cell growth factors. It is also known to exert potent immunosuppressive effects on a variety of immunological cell types, including inhibition of the proliferation of B lymphocytes (Petit-Koskas et al., Eur. J. Immunol., 18:111, 1988). It is. See also US Pat. No. 7,527,791.

The need to use TGFβ inhibitors, such as anti-TGFβ antibodies, to target a variety of diseases and medical conditions remains unmet. Additionally, there is a need to administer such TGFβ inhibitors in a manner that effectively treats a variety of diseases and medical conditions, including proliferative diseases, while maintaining ease of administration.

A method of treating a proliferative disease in a subject in need thereof, comprising administering to the subject a TGFβ antibody and at least one additional therapeutic agent, wherein the TGFβ antibody is administered at a dose of about 1400 to about 2100 mg. Disclosed herein are methods in which the patient is administered once every two, three, or four weeks. In some embodiments, the TGFβ antibody is administered at about 1400 mg once every two weeks. In some embodiments, the TGFβ antibody is administered at about 2100 mg once every two weeks. In some embodiments, the TGFβ antibody is administered at about 2100 mg once every three weeks. In some embodiments, the TGFβ antibody is administered at about 45 mg/kg once every three weeks. In some embodiments, the TGFβ antibody is administered at about 30 mg/kg once every three weeks. In some embodiments, the TGFβ antibody is administered at about 20 mg/kg once every three weeks.

The TGFβ antibody can include the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. The TGFβ antibody may comprise a heavy chain variable region and a light chain variable region shown in amino acid sequences SEQ ID NO: 7 and 8, respectively. A TGFβ antibody can include a heavy chain and a light chain as shown in the amino acid sequences of SEQ ID NO: 9 and 10, respectively.

In some embodiments, the TGFβ antibody is a monoclonal antibody. In some embodiments, the TGFβ antibody is a fully human antibody.

In some embodiments, the TGFβ inhibitor is administered over about 30 minutes.

In some embodiments, the additional therapeutic agent is a PD-1 inhibitor, gemcitabine, nab-paclitaxel, folinic acid (leucovorin calcium or levoleucovorin), fluorouracil (5-FU), oxaliplatin (eloxatin eloxatin)), bevacizumab, or irinotecan.

For example, in some embodiments, the additional therapeutic agent includes a PD-1 inhibitor. A PD-1 inhibitor can be an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is PDR001 (spartalizumab), BGB-A317 (tislelizumab), or BGB-108. For example, the anti-PD-1 antibody is tislelizumab. Tislelizumab may have a heavy chain of SEQ ID NO: 321. Tislelizumab may have a light chain of SEQ ID NO: 322. In some embodiments, the anti-PD-1 antibody is administered at 100 mg once per week. In some embodiments, the anti-PD-1 antibody is tislelizumab, administered at 300 mg per 28 day (ie, 4 week) cycle (Q4W). In some embodiments, the anti-PD-1 antibody is tislelizumab, administered at 200 mg per 21 day (ie, 3 week) cycle (Q3W).

In some embodiments, the additional therapeutic agent comprises gemcitabine. If gemcitabine is used, it may be administered at about 1000 mg/ ^m2 . In some embodiments, gemcitabine is administered on days 1, 8, and 15 of a 28-day cycle.

In some embodiments, the additional therapeutic agent comprises nab-paclitaxel. If nab-paclitaxel is used, it may be administered at about 125 mg/m ² . In some embodiments, nab-paclitaxel is administered on days 1, 8, and 15 of a 28-day cycle.

In some embodiments, the additional therapeutic agents include gemcitabine and nab-paclitaxel. In some embodiments, additional therapeutic agents include PD-inhibitors, gemcitabine and nab-paclitaxel.

In some embodiments, the additional therapeutic agent comprises bevacizumab. If bevacizumab is used, it may be administered at about 5 mg/ ^m2 . In some embodiments, bevacizumab is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent comprises 5-fluorouracil. If 5-fluorouracil is used, it may be administered at about 400 mg/m ² . In some embodiments, 5-fluorouracil may be administered at about 2400 mg/ ^m2 . In some embodiments, 5-fluorouracil is administered on days 1 and 15 of a 28 day cycle. In some embodiments, 5-fluorouracil is administered as an intravenous bolus. For example, 5-fluorouracil is administered as an intravenous bolus at 400 mg/ ^m2 on days 1 and 15 of each 28-day cycle, followed by a continuous intravenous infusion for 46 hours at 2400 mg/ ^m2. can be done.

In some embodiments, the additional therapeutic agent comprises leucovorin. If leucovorin is used, it may be administered at about 400 mg/ ^m2 . In some embodiments, leucovorin is administered on days 1 and 15 of a 28 day cycle. In some cases, leucovorin may be used instead of levoleucovorin. If levoleucovorin is used, it may be administered at 200 mg/ ^m2 . In some embodiments, levoleucovorin is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent comprises oxaliplatin. If oxaliplatin is used, it may be administered at about 85 mg/ ^m2 . In some embodiments, oxaliplatin is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent includes irinotecan. If irinotecan is used, it may be administered at about 180 mg/ ^m2 . In some embodiments, irinotecan is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, additional therapeutic agents include bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin. In some embodiments, additional therapeutic agents include PD-1 inhibitors, bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin. In some embodiments, additional therapeutic agents include bevacizumab, 5-fluorouracil, leucovorin, and irinotecan. In some embodiments, additional therapeutic agents include a PD-1 inhibitor, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan.

In some embodiments, the TGFβ antibody and additional therapeutic agent are administered to the subject in two or more cycles. In some embodiments, one cycle can be 28 days long. In some cases, cycles can be 21 days long.

The proliferative disease that can be treated with the TGFβ antibody and the additional therapeutic agent can be pancreatic cancer or colorectal cancer.

A method of treating pancreatic adenocarcinoma in a subject in need thereof, the method comprising administering to the subject 2100 mg of a TGFβ antibody, 1000 mg/m ² of gemcitabine, and 125 mg/m ² of nab-paclitaxel, wherein the TGFβ antibody is administered for a 28-day cycle. nab-paclitaxel was administered on days 1, 8, and 15 of the 28-day cycle, and the TGFβ antibody was administered in heavy chain CDR1 of SEQ ID NO: 1, 2, and 3, respectively. , CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively, are further disclosed herein.

In some embodiments, the method includes administering a PD-1 inhibitor to the patient. The PD-1 inhibitor can be spartalizumab or tislelizumab. If spartalizumab is administered, it may be administered at 400 mg once every four weeks. If tislelizumab is used, it may be administered at 100 mg per week of treatment. For example, tislelizumab may be administered at 400 mg once every four weeks. In some embodiments, the TGFβ antibody, gemcitabine and nab-paclitaxel (and optionally the PD-1 inhibitor) are administered to the subject in two or more cycles. In some embodiments, the TGFβ antibody, gemcitabine and nab-paclitaxel (and optionally a PD-1 inhibitor) are administered to the subject intravenously.

A method of treating colorectal cancer in a subject in need thereof, the method comprising: 2100 mg of TGFβ antibody, 5 mg/kg of bevacizumab, 400-2400 mg/m ² of 5-fluorouracil, 400 mg/m ² of leucovorin, and 85 mg/m ² of oxaliplatin. administering to the subject, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin are administered on days 1 and 15 of the 28-day cycle, and the TGFβ antibody comprises SEQ ID NO: 1, 2, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively, are disclosed herein.

In some embodiments, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin are administered to the subject in two or more cycles. In some embodiments, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin are administered to the subject intravenously. In some embodiments, 5-fluorouracil is administered as an intravenous bolus at 400 mg/m ² followed by a continuous intravenous infusion for 46 hours at 2400 mg/m ² .

In some embodiments, a method of treating colorectal cancer in a subject in need thereof, comprising: 2100 mg of TGFβ antibody, 5 mg/kg of bevacizumab, 400-2400 mg/m ² of 5-fluorouracil, 400 mg/m ² of leucovorin, and administering to the subject 180 mg/m ² of irinotecan, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan are administered on days 1 and 15 of the 28-day cycle, and the TGFβ antibody is SEQ ID NO. 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

In some embodiments, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan are administered to the subject in two or more cycles. In some embodiments, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan are administered to the subject intravenously. In some embodiments, 5-fluorouracil is administered as an intravenous bolus at 400 mg/m ² followed by a continuous intravenous infusion for 46 hours at 2400 mg/m ² .

In some embodiments, the TGFβ inhibitor is administered at the same time as the additional therapeutic agent. In some embodiments, the TGFβ inhibitor is administered before the additional therapeutic agent. In some embodiments, the TGFβ inhibitor is administered after the additional therapeutic agent.

In some embodiments, the TGFβ inhibitor and/or additional therapeutic agent is administered until remission.

In some embodiments, the additional therapeutic agent includes cyclophosphamide or topotecan. If cyclophosphamide is used, it may be administered at 250 mg/ ^m2 . In some embodiments, cyclophosphamide is administered for 5 days, such as 5 consecutive days. In some embodiments, the additional therapeutic agent comprises topotecan administered at 0.75 mg/ ^m2 . If topotecan is used, it may be administered for 5 days, eg, 5 consecutive days. In some embodiments, the proliferative disease is neuroblastoma.

In some embodiments, the additional therapeutic agent comprises gemcitabine. If gemcitabine is used, it may be administered at 675 mg/ ^m2 . In some embodiments, gemcitabine is administered for two days, such as days 1 and 8. In some embodiments, the proliferative disease is osteosarcoma.

In some embodiments, various combinations and doses can be used in subjects considered pediatric patients (eg, less than 18 years of age).

A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, comprising: (a) 2100 mg of a TGFβ antibody; (b) an intravenous bolus of 500 mg/m ² of 5-fluorouracil followed by a duration of 46 hours. (c) leucovorin (folinic acid) 400 mg/ ^{m 2 intravenously} or levoleucovorin 200 mg/m ² intravenously; (d) irinotecan 180 mg/m ² intravenously. , and (e) administering to the subject 5 mg/kg of bevacizumab, the TGFβ antibody is administered on day 1 (and optionally day 15) of the 28-day cycle, and 5-fluorouracil, leucovorin (or levoleucovorin) ), irinotecan, and bevacizumab were administered on days 1 and 15 of a 28-day cycle, and the TGFβ antibody was administered to heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and SEQ ID NO: 4. , 5, and 6, respectively, include light chain CDR1, CDR2, and CDR3.

A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, comprising: (a) 2100 mg of a TGFβ antibody; (b) an intravenous bolus of 400 mg/m ² of 5-fluorouracil followed by a duration of 46 hours. (c) leucovorin (folinic acid) 400 mg/ ^{m 2 intravenously} or levoleucovorin 200 mg/m ² intravenously; (d) irinotecan 180 mg/m ² intravenously. , and (e) administering to the subject 5 mg/kg of bevacizumab, the TGFβ antibody is administered on day 1 (and optionally day 15) of the 28-day cycle, and 5-fluorouracil, leucovorin (or levoleucovorin) ), irinotecan, and bevacizumab were administered on days 1 and 15 of a 28-day cycle, and the TGFβ antibody was administered to heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and SEQ ID NO: 4. , 5, and 6, respectively, include light chain CDR1, CDR2, and CDR3.

A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, comprising: (a) 2100 mg of a TGFβ antibody; (b) an intravenous bolus of 500 mg/m ² of 5-fluorouracil followed by a duration of 46 hours. (c) leucovorin (folinic acid) 400 mg/m ² intravenously or levoleucovorin 200 mg/m ² intravenously; (d ⁾ oxaliplatin 85 mg/m 2 intravenously. ² , and (e) administering to the subject 5 mg/kg of bevacizumab, the TGFβ antibody is administered on day 1 (and optionally day 15) of the 28-day cycle, and 5-fluorouracil, leucovorin (or levo leucovorin), oxaliplatin, and bevacizumab are administered on days 1 and 15 of a 28-day cycle, and the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 1, 2, and 3, respectively. Also disclosed herein are methods.

A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, comprising: (a) 2100 mg of a TGFβ antibody; (b) an intravenous bolus of 400 mg/m ² of 5-fluorouracil followed by a duration of 46 hours. (c) leucovorin (folinic acid) 400 mg/m ² intravenously or levoleucovorin 200 mg/m ² intravenously; (d ⁾ oxaliplatin 85 mg/m 2 intravenously. ² , and (e) administering to the subject 5 mg/kg of bevacizumab, the TGFβ antibody is administered on day 1 (and optionally day 15) of the 28-day cycle, and 5-fluorouracil, leucovorin (or levo leucovorin), oxaliplatin, and bevacizumab are administered on days 1 and 15 of a 28-day cycle, and the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 1, 2, and 3, respectively. Also disclosed herein are methods.

The method further includes administering to the patient a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is tislelizumab. Tislelizumab may have a heavy chain of SEQ ID NO: 321 and a light chain of SEQ ID NO: 322. In some embodiments, tislelizumab is administered intravenously at 300 mg on day 1 of each 28 day cycle.

In some embodiments, the TGFβ antibody, 5-fluorouracil, leucovorin (or levoleucovorin), oxaliplatin, irinotecan, bevacizumab, or PD-1 inhibitor is administered to the subject in two or more cycles.

A method of treating gastric cancer in a subject in need thereof, comprising: (a) 2100 mg of a TGFβ antibody, and (b) 200 mg of a PD-1 inhibitor in a Q3W cycle, in combination with (c) oxaliplatin on day 1. (d) 1000 mg/ ^{m 2} orally twice daily (days 1-14) of capecitabine twice daily (days 1-14) in a Q3W cycle; 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively, are also disclosed herein. The PD-1 inhibitor can be tislelizumab. In some embodiments, the TGFβ antibody is administered at a dose of 1400 mg or 2100 mg in Q2W cycles. In other embodiments, the PD-1 inhibitor is administered at a dose of 300 mg in Q4W cycles.

A method of treating gastric cancer in a subject in need thereof, comprising: (a) 2100 mg of a TGFβ antibody in a Q3W cycle, and (b) 200 mg of a PD-1 inhibitor in a Q3W cycle, in combination with (c) oxaliplatin. 85 mg/m ² intravenously (day 1); (d) leucovorin 400 mg/m ² or levoleucovorin 200 mg/m ² intravenously (day 1); (e) 5-fluorouracil 400 mg intravenously. / ^m2 (day 1) and (f) administering to the subject 1200 mg/ ^m2 by intravenous administration daily (days 1-2) in a Q2W cycle, wherein the TGFβ antibody is SEQ ID NO: 1, 2, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively, are also disclosed herein. The PD-1 inhibitor can be tislelizumab. In some embodiments, the TGFβ antibody is administered at a dose of 1400 mg or 2100 mg in Q2W cycles. In other embodiments, the PD-1 inhibitor is administered at a dose of 300 mg in Q4W cycles.

Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

The average concentration-time profile for each dose cohort of NIS793 during cycle 1 is shown. The average concentration-time profile for each dose cohort of NIS793 during cycle 3 is shown. Dosages and administration of various combinations of NIS793, spartalizumab or tislelizumab, gemcitabine, and/or nab-paclitaxel are shown. Spartalizumab or tislelizumab (400 mg) once every 4 weeks, combined with gemcitabine (1000 mg/m ² ) on days 1, 8, and 15, and nab-paclitaxel (125 mg/m ² ) on days 1, 8, and 15. NIS793 (2100 mg) will be administered to the patient once every two weeks (Figure 2). One cycle is 28 days.

The immune system is involved in early detection and destruction of cancer cells. Cancer cells can evade immune surveillance through various mechanisms, such as due to decreased immune recognition, increased resistance to attack by immune cells or an immunosuppressive tumor microenvironment (Mittal et al 2014). Some cancers produce the potent immunosuppressive cytokine TGFβ, which antagonizes cytotoxic lymphocytes and promotes the recruitment of suppressive immune cells that favor tumor growth and progression (Wojtowicz-Praga -2003, Teicher 2007, Yang et al 2010).

TGFβ belongs to a large family of structurally related cytokines that includes bone morphogenetic proteins (BMPs), growth differentiation factors, activins and inhibins. There are three isoforms of TGFβ ligand expressed in mammals: TGFβ1, 2, and 3. Under normal conditions, TGFβ maintains homeostasis and limits the growth of epithelial, endothelial, neural, and hematopoietic cell lineages through the induction of antiproliferative and apoptotic responses. Therefore, alterations in the TGFβ signaling pathway are thought to be involved in human diseases including cardiovascular disease, fibrosis, reproductive disorders, wound healing, and cancer (Wakefield and Hill 2013).

NIS793 is a fully human IgG2 human/mouse cross-reactive TGF-β neutralizing antibody. NIS793 acts at the ligand-receptor level. Compared to fresolimumab, a pan-TGFβ inhibitor that neutralizes all TGFβ isoforms, NIS793 antagonizes TGFβ1 and 2 more specifically and to a lesser extent TGFβ3.

To evade immune surveillance, cancer cells can additionally exploit immune checkpoint pathways that tightly control T cell activation, such as the PD-1/PD-L1 axis (Pardoll 2012). Thus, antagonizing TGFβ, alone or in combination with immune checkpoint blockade, may stimulate more potent anti-tumor immunity.

Definitions Additional terms are defined below and throughout this application.

As used herein, the articles "a" and "an" refer to one or more than one (e.g., at least one) of the grammatical referent of the article. .

The term "or" is used herein to mean and synonymously with the term "and/or" unless the context clearly dictates otherwise.

"About" and "approximately" mean an acceptable degree of error for the quantity being measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value or range of values. In some embodiments, when there is a reference to the term "about" in a number, the number is also intended to include the value of the number itself. For example, "about 10" includes, but is not limited to, the value 10. This also includes 10±2, 10±1, or 10±0.5.

"Combination" or "in combination with" may be used to imply that the therapies or therapeutic agents must be administered at the same time and/or must be formulated to be delivered together. Although not intended, these delivery methods are within the scope described herein. The therapeutic agents in the combination can be administered concurrently with, prior to, or subsequent to one or more other additional therapies or therapeutic agents. The therapeutic agents or therapeutic protocols may be administered in any order. Generally, each drug will be administered at a dose and/or according to a time schedule determined for that drug. Furthermore, it will be appreciated that the additional therapeutic agents utilized in the combination may be administered together in a single composition or separately in different compositions. The therapeutic agents may be administered in any order. The first agent and additional agents (eg, second, third agents) may be administered by the same route of administration or by different routes of administration. Generally, it is expected that the additional therapeutic agents utilized in combination will be utilized at levels not exceeding those at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than the levels utilized individually.

In some embodiments, the additional therapeutic agent is administered at a therapeutic dose or at a lower than therapeutic dose. In certain embodiments, the concentration of the second therapeutic agent necessary to achieve inhibition (e.g., growth inhibition) is such that the second therapeutic agent molecule) than when the second therapeutic agent (eg, anti-PD1 antibody molecule) is administered individually. In certain embodiments, the concentration of a first therapeutic agent required to achieve inhibition, e.g., growth inhibition, is such that when the first therapeutic agent is administered in combination with a second therapeutic agent: lower than when the first therapeutic agent is administered separately. In certain embodiments, in combination therapy, the concentration of the second therapeutic agent required to achieve inhibition, e.g., growth inhibition, is lower than the therapeutic dose of the second therapeutic agent as monotherapy; For example, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower. In certain embodiments, in combination therapy, the concentration of the first therapeutic agent required to achieve inhibition, e.g., growth inhibition, is lower than the therapeutic dose of the first therapeutic agent as monotherapy; For example, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower.

The term "inhibition", "inhibitor", or "antagonist" includes a reduction in certain parameters, eg, activity, of a given molecule, eg, an immune checkpoint inhibitor or a TGFβ inhibitor. For example, the term includes at least 5%, 10%, 20%, 30%, 40% or more inhibition of activity, eg, TGFβ, PD-1, or PD-L1 activity. Thus, inhibition may not be 100%.

The term "activation", "activator", or "agonist" includes an increase in certain parameters, eg, activity, of a given molecule, eg, costimulatory molecule. For example, the term includes an increase in activity, eg costimulatory activity, of at least 5%, 10%, 25%, 50%, 75% or more.

The term "anticancer effect" includes, but is not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or Refers to biological effects that can be manifested by various means, including amelioration of various physiological symptoms associated with cancerous pathology. "Anti-cancer effect" may also be manifested by the ability of peptides, polynucleotides, cells and antibodies in preventing the development of cancer in the first place.

The term "anti-tumor effect" may be manifested by a variety of means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell viability. Refers to biological effects.

The term "cancer" refers to a disease characterized by rapid, uncontrolled growth of abnormal cells (eg, a proliferative disease). Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein, including, but not limited to, solid tumors such as lung cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer. , and brain cancer, as well as hematological malignancies such as lymphoma and leukemia. The terms "tumor" and "cancer" are used interchangeably herein, eg, both terms encompass solid tumors and liquid tumors, such as diffuse tumors or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous as well as malignant cancers and tumors.

The term "antigen presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g. B cells, dendritic cells) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. cells, etc.). T cells can recognize such complexes using their T cell receptor (TCR). APCs process antigen and present it to T cells.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds a costimulatory ligand and thus mediates a costimulatory response by the T cell, including but not limited to proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activation molecules (SLAM proteins), and activated NK cell receptors. body, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS , ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2 Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55) , PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, Included are ligands that specifically bind to PAG/Cbp, CD19a, and CD83.

"Immune effector cell," or "effector cell," as the term is used herein, refers to a cell that is involved in promoting an immune response, eg, an immune effector response. Examples of immune effector cells include T cells, such as α/β and γ/δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. Examples include cells.

"Immune effector" or "effector" "function" or "response" as the term is used herein refers to, for example, a function or response of an immune effector cell that enhances or facilitates an immune attack on a target cell. Point. For example, an immune effector function or response refers to the property of a T cell or NK cell to promote killing of target cells or inhibition of their growth or proliferation. For T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "effector function" refers to a specialized function of a cell. For example, a T cell effector function can be a cytolytic activity or a helper activity, including secretion of cytokines.

As used herein, the terms "treat," "treatment," and "treating" refer to the progression, severity, or severity of a disorder, such as a proliferative disorder, resulting from the administration of one or more therapies. and/or a reduction or improvement in duration or improvement in one or more symptoms (preferably one or more recognizable symptoms) of the disorder. In specific embodiments, the terms "treat," "treatment," and "treating" refer to at least one measurable physical condition of the proliferative disorder, such as tumor growth, that is not necessarily perceptible to the patient. It refers to the improvement of physical parameters. In other embodiments, the terms "treat," "treatment," and "treating" refer to either physical, e.g., by stabilization of a discernible symptom, physiological, e.g., by stabilization of a physical parameter. , or both. In other embodiments, the terms "treat," "therapy," and "treating" refer to reducing or stabilizing tumor size or cancerous cell number.

The compositions, formulations, and methods of the invention include a specified sequence, or a sequence substantially identical or similar thereto, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity It includes polypeptides and nucleic acids having the sequence. In the context of amino acid sequences, the term "substantially identical" as used herein refers to the first and second amino acid sequences, such that the first and second amino acid sequences may have a common structural domain and/or a common functional activity. used to refer to the first amino acid that contains a sufficient number or a minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions for, the aligned amino acid residues in the amino acid sequences of Ru. For example, a reference sequence, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of a sequence provided herein. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

In the context of nucleotide sequences, the term "substantially identical" herein means that the first and second nucleotide sequences encode polypeptides with a common functional activity or share a common structural polypeptide domain. or a first nucleic acid sequence that contains a sufficient number or a minimum number of nucleotides that are identical to the aligned nucleotides in a second nucleic acid sequence so as to encode a common functional polypeptide activity. Ru. For example, a reference sequence, e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% of a sequence provided herein. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

The term "functional variant" refers to a variant that has a substantially identical amino acid sequence to, or is encoded by a substantially identical nucleotide sequence to, a naturally occurring sequence and that exhibits one or more of the activities of the naturally occurring sequence. refers to a polypeptide that has the ability to

Calculations of homology or sequence identity (these terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., one of the first and second amino acid or nucleic acid sequences is or gaps can be introduced in both and non-homologous sequences can be ignored for comparison purposes). In preferred embodiments, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical (as used herein) at that position. amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

The percent identity between two sequences is the number of identical positions they share, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. is a function of

Comparing sequences and determining percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined by the method of Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) with either a Blossum 62 matrix or a PAM250 matrix, and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3. , 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at www.gcg.com) using NWSgapdna. Determined using a CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and those to be used unless otherwise specified) is the Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

Percent identity between two amino acid or nucleotide sequences is determined by the E. coli as incorporated into the ALIGN program (version 2.0). Meyers and W. It can be determined using the algorithm of Miller ((1989) CABIOS, 4:11-17) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used, for example, as "query sequences" in performing searches of public databases to identify other family members or closely related sequences. Such a search is performed by Altschul, et al. (1990) J. Mol. Biol. 215:403-10 using the NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acids (molecules) of the invention. BLAST protein searches can be performed with the NBLAST program, score = 100, word length = 12. can be run with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to . To obtain gapped alignments for comparison purposes, see Altschul et al., (1997 ) Nucleic Acids Res. 25:3389-3402. When using the BLAST and Gapped BLAST programs, use the default parameters of each program (e.g., XBLAST and NBLAST). See www.ncbi.nlm.nih.gov.

As used herein, the term "hybridize under low stringency, moderate stringency, high stringency, or very high stringency conditions" describes hybridization and wash conditions. . For guidance on performing hybridization reactions, see Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. This reference describes aqueous and non-aqueous methods, both of which can be used. Specific hybridization conditions mentioned herein are: 1) low stringency hybridization conditions at about 45°C in 6x sodium chloride/sodium citrate (SSC) followed by 0 .2 washes at least 50°C in 2x SSC, 0.1% SDS (this wash temperature may be increased to 55°C for lower stringency conditions); 2) at about 45°C in 6x SSC; Moderate stringency hybridization conditions followed by one or more washes at 60°C in 0.2x SSC, 0.1% SDS; 3) high stringency hybridization at approximately 45°C in 6x SSC conditions, followed by one or more washes at 65°C in 0.2x SSC, 0.1% SDS; and preferably 4) very high stringency hybridization conditions: 0.5M sodium phosphate, 7% SDS. 65° C. in 0.2×SSC, 1% SDS followed by one or more washes at 65° C. in 0.2×SSC, 1% SDS. Extremely high stringency conditions (4) are the preferred conditions and should be used unless otherwise specified.

It is understood that the molecules of the invention may have additional conservative or non-essential amino acid substitutions that do not have a substantial effect on its function.

The term "amino acid" may include any molecule, whether natural or synthetic, that contains both amino and acidic functional groups and that is capable of being incorporated into polymers of naturally occurring amino acids. intended. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs thereof; amino acid analogs with variant side chains; and any stereoisomers of any one of the foregoing. As used herein, the term "amino acid" includes both D- or L-enantiomers and peptidomimetics.

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine).

The terms "polypeptide," "peptide," and "protein" (when single chain) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched and may include modified amino acids, which may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling moiety. . Polypeptides may be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or the product of synthetic procedures.

The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence," and "polynucleotide" are used interchangeably. These refer to nucleotides of any length in polymeric form, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be either single-stranded or double-stranded, and if single-stranded, it may be a coding strand or a non-coding strand (antisense strand). Polynucleotides can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation of label moieties. The nucleic acid may be a recombinant polynucleotide that does not occur in nature or is linked to another polynucleotide in a non-natural sequence, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin.

The term "isolated" as used herein refers to a material that has been removed from its original or native environment (eg, the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide found in a living animal is not isolated, but the same polynucleotide is separated by human intervention from some or all of the coexisting materials within a natural system. or the polypeptide has been isolated. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition, even if such a vector or composition isolated in that it is not part of the environment in which it is found in nature.

Various aspects of the invention are described in further detail below. Further definitions are provided throughout this specification.

TGF-β Inhibitors TGF-β belongs to a large family of structurally related cytokines that includes, for example, bone morphogenetic proteins (BMPs), growth differentiation factors, activins and inhibins. In some embodiments, the TGF-β inhibitors described herein include one or more isoforms of TGF-β (e.g., one of TGF-β1, TGF-β2, or TGF-β3). (one, two, or all) and/or inhibit it.

Throughout, transforming growth factor beta (also known as TGF-β, TGFβ, TGFb, or TGFbeta, used interchangeably herein) inhibitors (e.g., anti-TGF-β antibody molecules) are described, and This can be used in the methods described throughout.

In some embodiments, the TGF-β inhibitor is NIS793, fresolimumab, PF-06952229, or AVID200.

In some embodiments, the TGF-β inhibitor comprises a compound disclosed in NIS973, ie, WO 2012/167143. NIS793 is XOMA 089 or XPA. Also known as 42.089. NIS793 is a fully human monoclonal antibody that specifically binds and neutralizes TGF beta 1 and 2 ligands.

The heavy chain CDR1, CDR2, and CDR3 of NIS793 have the amino sequences of GGTFSSYAIS (SEQ ID NO: 1); GIIPIFGTANYAQKFQG (SEQ ID NO: 2); and GLWEVRALPSVY (SEQ ID NO: 3), respectively.

The light chain CDR1, CDR2, and CDR3 of NIS793 have the amino sequences of GANDIGSKSVH (SEQ ID NO: 4); EDIIRPS (SEQ ID NO: 5); and QVWDRDSDQYV (SEQ ID NO: 6), respectively.

The heavy chain variable region of NIS793 is: It has an amino acid sequence of WGQGTLVTVSS (SEQ ID NO: 7) (disclosed as SEQ ID NO: 6 in WO 2012/167143 pamphlet). The light chain variable region of NIS793 is SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQKAGQAPVLVVSEDIIRPSGIPERISGSNSGNTATLTISRVEAGDEADYYCQVWDRDSDQYVFGTGTKVTVLG( It has the amino acid sequence (SEQ ID NO: 8) (disclosed as SEQ ID NO: 8 in International Publication No. 2012/167143 pamphlet).

The heavy chain of NIS793 is

It has the amino acid sequence of The light chain of NIS793 is

It has the amino acid sequence of

NIS793 binds with high affinity to the human TGF-β isoform. In general, NIS793 binds TGF-β1 and TGF-β2 with high affinity and to a lesser extent TGF-β3. In the Biacore assay, the K _D of NIS793 for human TGF-β is 14.6 pM for TGF-β1, 67.3 pM for TGF-β2, and 948 pM for TGF-β3. Given its high affinity binding to all three TGF-β isoforms, in certain embodiments, NIS793 binds TGF-β1, 2 and It seems to be combined with 3. NIS793 cross-reacts with rodent and cynomolgus monkey TGF-β and exhibits functional activity in vitro and in vivo, making rodents and cynomolgus monkeys suitable species for toxicity studies.

In certain embodiments, TGF-β inhibitors are used to treat cancer (eg, pancreatic cancer, such as PDAC, or gastrointestinal cancer, such as colorectal cancer). In some embodiments, TGF-β inhibitors are used in combination with checkpoint inhibitors (eg, the inhibitors of PD1 described herein) to treat cancer.

In some embodiments, the TGF-β inhibitor (eg, NIS793) is administered at a dose higher than 15 mg/kg. For example, a TGF-β inhibitor is administered at a dose of 15.1 mg/kg to about 50 mg/kg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 16 mg/kg to about 50 mg/kg. In some embodiments, the TGF-β inhibitor is administered at a dose of 16 mg/kg to about 50 mg/kg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 20 mg/kg to about 40 mg/kg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 25 mg/kg to about 35 mg/kg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 20 mg/kg. In some embodiments, the TGFβ inhibitor is administered at a dose of about 30 mg/kg. In some embodiments, the TGFβ inhibitor is administered at a dose of about 45 mg/kg.

In some embodiments, the TGF-β inhibitor (eg, NIS793) is administered in a fixed dose. For example, in some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg to about 1600 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1100 mg to about 1500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1200 to about 1400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1300 mg to about 1400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1300 mg to about 1500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1300 mg to about 1600 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1200 mg to about 1500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1200 mg to about 1600 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1400 mg to about 1500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1400 mg to about 1600 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1100 mg to about 1600 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of 1100 mg to about 1400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1100 mg to about 1300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1100 mg to about 1200 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg to about 1500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg to about 1400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg to about 1300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg to about 1200 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg to about 1100 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1000 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1100 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1200 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1600 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1200 mg to about 2100 mg.

In some embodiments, the TGF-β inhibitor is administered at a dose of about 2000 mg to about 2500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2000 mg to about 2400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1900 mg to about 2300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 1900 mg to about 2200 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2000 mg to about 2100 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2100 mg to about 2500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2100 to about 2400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2100 to about 2300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2100 to about 2200 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2200 to about 2500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2200 to about 2400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2200 to about 2300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2300 mg to about 2500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2300 mg to about 2400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2400 mg to about 2500 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2000 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2100 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2200 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2300 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2400 mg. In some embodiments, the TGF-β inhibitor is administered at a dose of about 2500 mg.

In some embodiments, the TGF-β inhibitor is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the TGF-β inhibitor is administered once a week. In some embodiments, the TGF-β inhibitor is administered once every two weeks. In some embodiments, the TGF-β inhibitor is administered once every three weeks. In some embodiments, the TGF-β inhibitor is administered once four three weeks.

In some embodiments, the TGF-β inhibitor is administered intravenously.

In some embodiments, the TGF-β inhibitor is administered over about 20 minutes to about 40 minutes. For example, TGF-β inhibitors are administered over about 30 minutes. In some embodiments, the TGF-β inhibitor is administered over about 1 hour. In some embodiments, the TGF-β inhibitor is administered over about 2 hours. In some embodiments, the TGF-β inhibitor is administered over about 3 hours. In some embodiments, the TGF-β inhibitor is administered over about 4 hours. In some embodiments, the TGF-β inhibitor is administered over about 5 hours. In some embodiments, the TGF-β inhibitor is administered over about 6 hours.

In some embodiments, the TGFβ inhibitor (eg, NIS793) is administered intravenously once every two weeks at a dose of about 1300 to about 1500 mg (eg, about 1400 mg). In some embodiments, the TGFβ inhibitor (eg, NIS793) is administered intravenously once every two weeks at a dose of about 2000 to about 2200 mg (eg, about 2100 mg). In some embodiments, the TGFβ inhibitor (eg, NIS793) is administered intravenously at a dose of about 2000 to about 2200 mg (eg, about 2100 mg) once every three weeks.

In some embodiments, the TGF-β inhibitor (e.g., NIS793) is administered intravenously for about 20 minutes to about 40 minutes (e.g., about 30 minutes) at a dose of about 1300 mg to about 1500 mg (e.g., about 1400 mg). minutes) and is administered once every two weeks. In some embodiments, the TGF-β inhibitor (e.g., NIS793) is administered intravenously for about 20 minutes to about 40 minutes (e.g., about 30 minutes) at a dose of about 2000 mg to about 2200 mg (e.g., about 2100 mg). minutes) and is administered once every two weeks. In some embodiments, the TGF-β inhibitor (e.g., NIS793) is administered intravenously for about 20 minutes to about 40 minutes (e.g., about 30 minutes) at a dose of about 2000 mg to about 2200 mg (e.g., about 2100 mg). It is administered once every 3 weeks over a period of 30 minutes.

In some embodiments, the TGFβ inhibitor (eg, NIS793) is administered intravenously once every three weeks at a dose of about 40 to about 50 mg/kg (eg, about 45 mg/kg). In some embodiments, the TGFβ inhibitor (eg, NIS793) is administered intravenously once every three weeks at a dose of about 25 to about 35 mg/kg (eg, about 30 mg/kg). In some embodiments, the TGFβ inhibitor (eg, NIS793) is administered intravenously once every three weeks at a dose of about 15 to about 25 mg/kg (eg, about 20 mg/kg).

In some embodiments, the methods described herein can further include one or more other therapeutic agents, procedures, or modalities. In some embodiments, the TGF-β inhibitor (e.g., NIS793) is combined with a PD1 inhibitor (e.g., an anti-PD1 antibody molecule) and/or a PD-L inhibitor (PD-L1 and/or PD-L2). Administered in combination. In one embodiment, the methods described herein can include administering an inhibitor of the suppressive (or immune checkpoint) molecules PD-1, PD-L1, PD-L2, and/or TGFβ. . In one embodiment, the inhibitor is an antibody or antibody fragment that binds PD-1, PD-L1, PD-L2, and/or TGFβ.

Alternatively, or in combination with the foregoing methods, the methods described herein include immunomodulatory agents (e.g., activators of costimulatory molecules or inhibitory molecules, e.g., inhibitors of immune checkpoint molecules); It may be administered or used in conjunction with one or more of vaccines, such as therapeutic cancer vaccines; or other forms of cellular immunotherapy.

In certain embodiments, the methods described herein are administered or used in conjunction with co-stimulatory or inhibitory molecules, such as co-inhibitory ligands or receptor modulators.

Other Exemplary TGF-β Inhibitors In some embodiments, the TGF-β inhibitor comprises fresolimumab (CAS Registration Number: 948564-73-6). Fresolimumab is also known as GC1008. Fresolimumab is a human monoclonal antibody that binds to and inhibits TGF beta isoforms 1, 2 and 3.

The heavy chain of fresolimumab is

It has the amino acid sequence of

The light chain of fresolimumab is

It has the amino acid sequence of

Fresolimumab is described, for example, in WO 2006/086469 and US Patent Nos. 8,383,780 and 8,591,901.

In some embodiments, the TGF-β inhibitor is PF-06952229. PF-06952229 is an inhibitor of TGF-βR1, preventing signaling through this receptor and TGF-βR1-mediated immunosuppression, thereby enhancing anti-tumor immune responses. For PF-06952229, see, for example, Yano et al. Immunology 2019; 157(3) 232-47.

In some embodiments, the TGF-β inhibitor is AVID200. AVID200 is a TGF-β receptor ectodomain-IgG Fc fusion protein that selectively targets and neutralizes TGF-β isoforms 1 and 3. Regarding AVID200, see, for example, O'Connor-McCourt, MD et al. Can. Res. 2018;78(13).

PD-1 Inhibitors PD-1 is a CD28/CTLA-4 family member expressed, for example, on activated CD4+ and CD8+ T cells, Tregs, and B cells. It negatively regulates effector T cell signaling and function. PD-1 is induced on tumor-infiltrating T cells and can result in functional depletion or dysfunction (Keir et al. (2008) Annu. Rev. Immunol. 26:677-704; Pardoll et al. (2012) Nat Rev Cancer 12(4):252-64). PD-1 delivers co-inhibitory signals when bound to either of its two ligands, programmed death ligand 1 (PD-L1) or programmed death ligand 2 (PD-L2). PD-L1 is expressed on numerous cell types, including T cells, natural killer (NK) cells, macrophages, dendritic cells (DCs), B cells, epithelial cells, vascular endothelial cells, and many types of tumors. . High expression of PD-L1 in mouse and human tumors has been associated with poor clinical outcomes in various cancers (Keir et al. (2008) Annu. Rev. Immunol. 26:677-704; Pardoll et al. (2012) Nat Rev Cancer 12(4):252-64). PD-L2 is expressed on dendritic cells, macrophages, and some tumors. Blockade of the PD-1 pathway has been validated preclinically and clinically for cancer immunotherapy. Both preclinical and clinical studies have demonstrated that anti-PD-1 blockade can restore effector T cell activity, resulting in robust anti-tumor responses. For example, blockade of the PD-1 pathway can restore depleted/dysfunctional effector T cell function (e.g., proliferation, IFN-γ secretion, or cytolytic function) and/or inhibit Treg cell function. (Keir et al. (2008) Annu. Rev. Immunol. 26:677-704; Pardoll et al. (2012) Nat Rev Cancer 12(4):252-64). Blocking the PD-1 pathway can be achieved with PD-1, PD-L1 and/or PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, or oligopeptides.

As used herein, the term "programmed death 1" or "PD-1" includes isoforms, mammalian, e.g., human PD-1, species homologs of human PD-1, and common species with PD-1. Analogs containing at least one epitope are included. The amino acid sequence of PD-1, eg, human PD-1, is described, eg, by Shinohara T et al. (1994) Genomics 23(3):704-6; Finger LR, et al. Gene (1997) 197(1-2):177-87, etc., as known in the art.

In certain embodiments, a TGFβ inhibitor described herein is administered in conjunction with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is spartalizumab (PDR001, Novartis), nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co), pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron) , TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (also known as tislelizumab) (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune) is .

Exemplary PD-1 Inhibitors In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is described in U.S. Patent Application Publication No. 2015/0210769, entitled "Antibody Molecules to PD-1 and Uses Thereof," published July 30, 2015. It is an anti-PD-1 antibody molecule. In one embodiment, the anti-PD-1 inhibitor is spartalizumab, also known as PDR001. In another embodiment, the anti-PD-1 inhibitor is tislelizumab, also known as BGB-A317.

In one embodiment, anti-PD-1 antibody molecules are derived from heavy and light chain variable regions comprising the amino acid sequences set forth in Table 1, or the amino acid sequences encoded by the nucleotide sequences set forth in Table 1 (e.g., at least 1, 2, 3, 4, 5 or 6 complementarity determining regions (CDRs) (from the heavy chain and light chain variable region sequences of BAP049-clone-E or BAP049-clone-B disclosed in 1. including all CDRs). In some embodiments, the CDRs follow the Kabat definition (eg, as shown in Table 1). In some embodiments, the CDRs follow the Chothia definition (eg, as shown in Table 1). In some embodiments, the CDRs follow the combination of both Kabat and Chothia CDR definitions (eg, as shown in Table 1). In one embodiment, the Kabat and Chothia combination CDR of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 13). In one embodiment, one or more of the CDRs (or all CDRs collectively) are 1, 2, Having 3, 4, 5, 6 or more changes, such as amino acid substitutions (eg, conservative amino acid substitutions) or deletions.

In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain variable region comprising a VHCDR1 amino acid sequence of SEQ ID NO: 14, a VHCDR2 amino acid sequence of SEQ ID NO: 15, and a VHCDR3 amino acid sequence of SEQ ID NO: 16, each disclosed in Table 1. (VH); and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 23, the VLCDR2 amino acid sequence of SEQ ID NO: 24, and the VLCDR3 amino acid sequence of SEQ ID NO: 25.

In one embodiment, the antibody molecules are VHCDR1 encoded by the nucleotide sequence SEQ ID NO: 37, VHCDR2 encoded by the nucleotide sequence SEQ ID NO: 38, and VHCDR2 encoded by the nucleotide sequence SEQ ID NO: 39, each disclosed in Table 1. a VH comprising VHCDR3; and a VL comprising VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 42, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 43, and VLCDR3 encoded by the nucleotide sequence of SEQ ID NO: 44.

In one embodiment, the anti-PD-1 antibody molecule has the amino acid sequence of SEQ ID NO: 19, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes VHs that have amino acid sequences of 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity. In one embodiment, the anti-PD-1 antibody molecule has the amino acid sequence of SEQ ID NO: 33, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes VLs that have amino acid sequences of 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity. In one embodiment, the anti-PD-1 antibody molecule has the amino acid sequence of SEQ ID NO: 29, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes VLs that have amino acid sequences of 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 33. In one embodiment, the anti-PD-1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 33.

In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 20, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, or 99% or more identical nucleotide sequences. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 34 or 30, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes VLs encoded by nucleotide sequences that are 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 20 and a VL encoded by the nucleotide sequence of SEQ ID NO: 34 or 30.

In one embodiment, the anti-PD-1 antibody molecule comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 21. Including chains. In one embodiment, the anti-PD-1 antibody molecule has the amino acid sequence of SEQ ID NO: 35, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes light chains that have amino acid sequences that are 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical. In one embodiment, the anti-PD-1 antibody molecule has the amino acid sequence of SEQ ID NO: 31, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes light chains that have amino acid sequences that are 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 31.

In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 22, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, or 99% or more. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 36 or 32, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Includes light chains encoded by nucleotide sequences that are 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 22 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 36 or 32.

In another embodiment, the anti-PD-1 antibody molecule, eg, tislelizumab, comprises the following heavy and/or light chains: VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody comprising SEQ ID NO: 321. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody comprising SEQ ID NO: 322. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody comprising SEQ ID NOs: 321 and 322. In some embodiments, the PD-1 inhibitor comprises the HCDR and LCDR of tislelizumab set forth in SEQ ID NOs: 325-330.

The antibody molecules described herein may be made by vectors, host cells, and methods described in US Patent Application Publication No. 2015/0210769.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered at a fixed dose of about 100 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 100 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 100 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 100 mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 100 mg to about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 400 mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 400 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 500 mg to about 600 mg.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab) is administered at a fixed dose of about 100 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 800 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 1000 mg.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered once every 10 days. In some embodiments, the PD-1 inhibitor is administered once every nine weeks. In some embodiments, the PD-1 inhibitor is administered once every eight weeks. In some embodiments, the PD-1 inhibitor is administered once every 7 weeks. In some embodiments, the PD-1 inhibitor is administered once every 6 weeks. In some embodiments, the PD-1 inhibitor is administered once every five weeks. In some embodiments, the PD-1 inhibitor is administered once every four weeks. In some embodiments, the PD-1 inhibitor is administered once every three weeks. In some embodiments, the PD-1 inhibitor is administered once every two weeks. In some embodiments, the PD-1 inhibitor is administered once a week.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered intravenously.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered over about 20 to 40 minutes (eg, about 30 minutes). In some embodiments, the PD-1 inhibitor is administered over about 30 minutes. In some embodiments, the PD-1 inhibitor is administered over about 1 hour. In some embodiments, the PD-1 inhibitor is administered over about 2 hours. In some embodiments, the PD-1 inhibitor is administered over about 3 hours. In some embodiments, the PD-1 inhibitor is administered over about 4 hours. In some embodiments, the PD-1 inhibitor is administered over about 5 hours. In some embodiments, the PD-1 inhibitor is administered over about 6 hours.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered intravenously at a dose of about 300 to about 500 mg (eg, about 400 mg) once every four weeks. In some embodiments, the PD-1 inhibitor is administered intravenously at a dose of about 200 to about 400 mg (eg, about 300 mg) once every three weeks. In some embodiments, spartalizumab or tislelizumab is administered intravenously at a dose of 400 mg once every four weeks. In some embodiments, spartalizumab or tislelizumab is administered intravenously at a dose of 300 mg once every four weeks. In some embodiments, spartalizumab or tislelizumab is administered intravenously at a dose of 300 mg once every three weeks. In some embodiments, spartalizumab or tislelizumab is administered intravenously at a dose of 200 mg once every three weeks.

In some embodiments, the PD-1 inhibitor (e.g., spartalizumab or tislelizumab) is administered at a dosage of about 300 to about 500 mg over a period of about 20 to about 40 minutes (e.g., about 30 minutes) once every two weeks. (eg, about 400 mg) intravenously. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 to about 400 mg (e.g., about 300 mg) over a period of about 20 to about 40 minutes (e.g., about 30 minutes) once every three weeks. Administered intravenously. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 to about 400 mg (e.g., about 300 mg) over a period of about 20 to about 40 minutes (e.g., about 30 minutes) once every four weeks. Administered intravenously. In some embodiments, the PD-1 inhibitor is administered at a dose of about 100 to about 300 mg (e.g., about 200 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes) once every three weeks. It is administered intravenously.

In some embodiments, the PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered at a dose of about 100 mg/week. For example, if a 10 week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 1000 mg. If a 9-week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 900 mg. If the 8-week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 800 mg. If the 7-week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 700 mg. If a 6 week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 600 mg. If a 5 week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 500 mg. If a 4-week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 400 mg. If a 3-week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 300 mg. If a two-week dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 200 mg. If a weekly dose is administered to a patient, then the PD-1 inhibitor (eg, spartalizumab or tislelizumab) may be administered at 100 mg.

For example, if an anti-PD-1 antibody such as tislelizumab is used, it may be administered as an intravenous infusion once every three weeks at a dose of 200 mg. Alternatively, tislelizumab may be administered as an intravenous infusion once every four weeks at a dose of 300 mg. If an anti-PD-1 antibody such as spartalizumab or tislelizumab is used, it may be administered at a dose of 300 mg as an intravenous infusion once every three weeks. Alternatively, spartalizumab or tislelizumab may be administered as an intravenous infusion once every four weeks at a dose of 400 mg.

In some embodiments, a PD-1 inhibitor (eg, spartalizumab or tislelizumab) is administered in conjunction with a TGFβ inhibitor (eg, NIS793).

Tislelizumab is a human programmed cell death therapy approved by the Chinese Food and Drug Administration (CFDA) for various indications including Hodgkin lymphoma, urothelial carcinoma, hepatocellular carcinoma, and squamous, non-squamous, non-small cell lung cancer. A humanized IgG monoclonal antibody against PD-1 (PD-1) and BLA (Biologics License Application) against esophageal squamous cell carcinoma (ESCC) has been approved by the FDA.

In specific embodiments, tislelizumab may be administered in conjunction with a TGFβ antibody (eg, NIS793) and/or in conjunction with any of the disclosed chemotherapeutic agents. The amino acid sequence of tislelizumab is disclosed above and includes SEQ ID NOs: 321-330.

In some embodiments, tislelizumab is administered to the patient (in need thereof) at dosages disclosed throughout. For example, tislelizumab can be administered as a fixed dose of about 200-400 mg. More specifically, tislelizumab in these specific embodiments may be administered to a patient at a dose of 300 mg.

When tislelizumab is used, the frequency of dosing can be as described throughout. For example, a frequency of once every three weeks (Q3W) or once every four weeks (Q4W) may be used.

In one specific example, tislelizumab can be administered to a subject at a dose of 300 mg once every four weeks. In another specific example, tislelizumab can be administered to a subject at a dose of 200 mg once every three weeks.

Other Exemplary PD-1 Inhibitors In one embodiment, the anti-PD-1 antibody molecule is nivolumab (Bristol-Myers Squibb), also known as MDX-1106, MDX-1106-04, ONO-4538, BMS-936558, or OPDIVO (registered trademark). Nivolumab (clone 5C4) and other anti-PD-1 antibodies are disclosed in US Pat. No. 8,008,449 and WO 2006/121168. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of nivolumab, the heavy chain or light chain variable region, e.g., as disclosed in Table 2. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is pembrolizumab (Merck & Co), also known as lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®. For information on pembrolizumab and other anti-PD-1 antibodies, see Hamid, O.; et al. (2013) New England Journal of Medicine 369(2):134-44, US Pat. No. 8,354,509, and WO 2009/114335. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of pembrolizumab, the heavy chain or light chain variable region, e.g., as disclosed in Table 2. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is pidilizumab (CureTech), also known as CT-011. For pidilizumab and other anti-PD-1 antibodies, see Rosenblatt, J. et al. (2011) J Immunotherapy 34(5):409-18, U.S. Patent No. 7,695,715, U.S. Patent No. 7,332,582, and U.S. Patent No. 8,686,119 has been disclosed. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of pidilizumab, the heavy chain or light chain variable region, e.g., as disclosed in Table 2. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in US Pat. No. 9,205,148 and WO 2012/145493. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences of MEDI0680 (or all CDR sequences collectively), a heavy or light chain variable region sequence, or a heavy or light chain sequence. include.

In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (Regeneron). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences of REGN2810 (or all CDR sequences collectively), a heavy chain or light chain variable region sequence, or a heavy or light chain sequence. include.

In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of PF-06801591, the heavy chain or light chain variable region sequence, or the heavy or light chain Contains arrays.

In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 or BGB-108 (Beigene). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences of BGB-A317 or BGB-108 (or all CDR sequences taken together), the heavy chain or light chain variable region sequences, or the heavy chain chain or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences of INCSHR 1210 (or all CDR sequences collectively), a heavy or light chain variable region sequence, or a heavy or light chain sequence. include.

In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of TSR-042, the heavy or light chain variable region sequences, or the heavy or light chain Contains arrays.

Further known anti-PD-1 antibodies include, for example, International Publication No. 2015/112800 pamphlet, International Publication No. 2016/092419 pamphlet, International Publication No. 2015/085847 pamphlet, International Publication No. 2014/179664 pamphlet, International Publication No. 2014/194302 pamphlet, International Publication No. 2014/209804 pamphlet, International Publication No. 2015/200119 pamphlet, US Patent No. 8,735,553, US Patent No. 7,488,802 , US Pat. No. 8,927,697, US Pat. No. 8,993,731, and US Pat. No. 9,102,727.

In one embodiment, the anti-PD-1 antibody is an antibody that competes for binding with and/or binds to the same epitope on PD-1 as one of the anti-PD-1 antibodies described herein. be.

In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, eg, as described in US Pat. No. 8,907,053. In one embodiment, the PD-1 inhibitor is an immunoadhesin, such as the extracellular portion or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In one embodiment, the PD-1 inhibitor is an immunoadhesin comprising AMP-224 (e.g. B7-DCIg as disclosed in WO 2010/027827 and WO 2011/066342). (Amplimune)).

PD-L1 Inhibitors In certain embodiments, the described methods further include administering a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is FAZ053 (Novartis), atezolizumab (Genentech/Roche), avelumab (Merck Serono and Pfizer), durvalumab (MedImmune/AstraZeneca), or BMS-93655 9 (Bristol-Myers Squibb ).

Exemplary PD-L1 Inhibitors In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is described in U.S. Patent Application Publication No. 2016/0108123, entitled "Antibody Molecules to PD-L1 and Uses Thereof," published April 21, 2016. An anti-PD-L1 antibody molecule as disclosed.

In one embodiment, anti-PD-L1 antibody molecules are derived from heavy and light chain variable regions comprising the amino acid sequences set forth in Table 3, or the amino acid sequences encoded by the nucleotide sequences set forth in Table 3 (e.g., at least 1, 2, 3, 4, 5 or 6 complementarity determining regions (CDRs) (or collectively from the heavy chain and light chain variable region sequences of BAP058-clone O or BAP058-clone N disclosed in Contains all CDRs). In some embodiments, the CDRs follow the Kabat definition (eg, as shown in Table 3). In some embodiments, the CDRs follow the Chothia definition (eg, as shown in Table 3). In some embodiments, the CDRs follow the combination of both Kabat and Chothia CDR definitions (eg, as shown in Table 3). In one embodiment, the Kabat and Chothia combination CDR of VH CDR1 comprises the amino acid sequence GYTFTSYWMY (SEQ ID NO: 100). In one embodiment, one or more of the CDRs (or all CDRs collectively) are 1, 2, Having 3, 4, 5, 6 or more changes, such as amino acid substitutions (eg, conservative amino acid substitutions) or deletions.

In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain variable region comprising a VHCDR1 amino acid sequence of SEQ ID NO: 54, a VHCDR2 amino acid sequence of SEQ ID NO: 55, and a VHCDR3 amino acid sequence of SEQ ID NO: 56, each disclosed in Table 3. (VH); and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 62, the VLCDR2 amino acid sequence of SEQ ID NO: 63, and the VLCDR3 amino acid sequence of SEQ ID NO: 64.

In one embodiment, the anti-PD-L1 antibody molecules are VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 81, VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 82, and nucleotide sequence of SEQ ID NO: 83, respectively disclosed in Table 3. a VH comprising VHCDR3 encoded by the sequence; VLCDR1 encoded by the nucleotide sequence SEQ ID NO: 86; VLCDR2 encoded by the nucleotide sequence SEQ ID NO: 87; and VL comprising VLCDR3 encoded by the nucleotide sequence SEQ ID NO: 88. including.

In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 59, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, A VH comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity . In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 69, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Comprising a VL comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity . In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 73, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, A VH comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity . In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 77, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, Comprising a VL comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity . In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 59 and a VL comprising the amino acid sequence of SEQ ID NO: 69. In one embodiment, the anti-PD-L1 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 77.

In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 60, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 70, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 74, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 78, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 60 and a VL encoded by the nucleotide sequence of SEQ ID NO: 70. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 72 and a VL encoded by the nucleotide sequence of SEQ ID NO: 78.

In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 61, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, a heavy chain comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity; include. In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 71, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, a light chain comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity; include. In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 75, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, a heavy chain comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity; include. In one embodiment, the anti-PD-L1 antibody molecule has the amino acid sequence of SEQ ID NO: 79, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, a light chain comprising an amino acid sequence of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity; include. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO: 71. In one embodiment, the anti-PD-L1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 75 and a light chain comprising the amino acid sequence of SEQ ID NO: 79.

In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 68, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 72, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO: 76, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. In one embodiment, the antibody molecule has the nucleotide sequence of SEQ ID NO:80, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% with SEQ ID NO:80. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 68 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 72. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 76 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 80.

The antibody molecules described herein may be made by vectors, host cells, and methods described in US Patent Application Publication No. 2016/0108123.

Other Exemplary PD-L1 Inhibitors In one embodiment, the anti-PD-L1 antibody molecule is atezolizumab (Genentech/Roche), also known as MPDL3280A, RG7446, RO5541267, YW243.55. S70, or TECENTRIQ (trademark). Atezolizumab and other anti-PD-L1 antibodies are disclosed in US Pat. No. 8,217,149. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of atezolizumab, the heavy chain or the light chain variable region, e.g., as disclosed in Table 4. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-PD-L1 antibody molecule is avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO 2013/079174 pamphlet. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of avelumab, the heavy chain or light chain variable region, e.g., as disclosed in Table 4. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-PD-L1 antibody molecule is durvalumab (MedImmune/AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in US Pat. No. 8,779,108. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of durvalumab, the heavy chain or light chain variable region, e.g., as disclosed in Table 4. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), MDX-1105, or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in U.S. Pat. No. 7,943,743 and WO 2015/081158. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the CDR sequences (or collectively all of the CDR sequences), heavy or light chain variable region sequences, or heavy or light chain sequences of BMS-936559, e.g., as disclosed in Table 4.

Further known anti-PD-L1 antibodies include, for example, International Publication No. 2015/181342 pamphlet, International Publication No. 2014/100079 pamphlet, International Publication No. 2016/000619 pamphlet, International Publication No. 2014/022758 pamphlet, International Publication No. 2014/055897 pamphlet, International Publication No. 2015/061668 pamphlet, International Publication No. 2013/079174 pamphlet, International Publication No. 2012/145493 pamphlet, International Publication No. 2015/112805 pamphlet, International Publication No. 2015/ 109124 pamphlet, International Publication No. 2015/195163 pamphlet, US Patent No. 8,168,179, US Patent No. 8,552,154, US Patent No. 8,460,927, and Examples include those described in US Pat. No. 9,175,082.

In one embodiment, the anti-PD-L1 antibody is an antibody that competes for binding with and/or binds to the same epitope on PD-L1 as one of the anti-PD-L1 antibodies described herein. be.

Antibodies and Antibody-Like Molecules As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, that includes at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes, for example, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region). In certain embodiments, the antibody molecule comprises a full-length antibody or full-length immunoglobulin chain. In certain embodiments, the antibody molecule comprises a full-length antibody, or an antigen-binding or functional fragment of a full-length immunoglobulin chain. In certain embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first of the plurality of immunoglobulin variable domain sequences is associated with a first epitope. a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope (e.g., a second target); has. In certain embodiments, the multispecific antibody molecule is a bispecific antibody molecule.

In certain embodiments, the antibody molecule is a monospecific antibody molecule and binds a single epitope (eg, a single target, such as TGFβ-like NIS793). For example, a monospecific antibody molecule may have multiple immunoglobulin variable domain sequences, each of which binds the same epitope.

In certain embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope (e.g., a second target); has. In certain embodiments, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In certain embodiments, the first and second epitopes overlap. In certain embodiments, the first and second epitopes are non-overlapping. In certain embodiments, the first and second epitopes are on different antigens, eg, different proteins (or different subunits of a multimeric protein). In certain embodiments, the multispecific antibody molecule includes a third, fourth, or fifth immunoglobulin variable domain. In certain embodiments, the multispecific antibody molecule is a bispecific, trispecific, or tetraspecific antibody molecule.

In certain embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for two or fewer antigens. A bispecific antibody molecule comprises a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. It is characterized by In certain embodiments, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In certain embodiments, the first and second epitopes overlap. In certain embodiments, the first and second epitopes are non-overlapping. In certain embodiments, the first and second epitopes are on different antigens, eg, different proteins (or different subunits of a multimeric protein). In certain embodiments, a bispecific antibody molecule has a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and that have binding specificity for a second epitope. A heavy chain variable domain sequence and a light chain variable domain sequence. In certain embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises a half-antibody, or a fragment thereof, that has binding specificity for a first epitope and a half-antibody, or a fragment thereof, that has binding specificity for a second epitope. including fragments. In certain embodiments, the bispecific antibody molecule comprises an scFv, or fragment thereof, that has binding specificity for a first epitope and an scFv, or fragment thereof, that has binding specificity for a second epitope. including. In certain embodiments, the first epitope is on TGFβ (1, 2, and/or 3) and the second epitope is on PD-1 (or PD-L1 or PD-L2).

Protocols for making multispecific (e.g., bispecific or trispecific) or heterodimeric antibody molecules are known in the art; for example, without limitation, U.S. Pat. No. 5,731,168 WO 09/089004 pamphlet, WO 06/106905 pamphlet and WO 2010/129304 pamphlet. Electrostatic steering Fc pairing; Strand exchange engineering domain (SEED) heterodimer formation as described for example in WO 07/110205; WO 08/119353, WO 2011/131746 Fab arm exchange, e.g. as described in US Pat. No. 4,433,059, and WO 2013/060867; double antibody conjugates by antibody cross-linking to create bispecific structures using heterobifunctional reagents with groups; as described, e.g., in U.S. Pat. No. 4,444,878; Bispecific antibody determinants created by recombining half antibodies (heavy chain-light chain pairs or Fabs) of different antibodies through reduction and oxidation cycles of the disulfide bond between the two heavy chains; US Pat. , 273,743; e.g., three Fab' fragments crosslinked by sulfhydryl reactive groups; Biosynthetic binding proteins as described, e.g. pairs of scFv cross-linked at the C-terminal tails, preferably by disulfide- or amine-reactive chemical cross-linking; e.g. as described in U.S. Pat. No. 5,582,996 Bifunctional antibodies as described, e.g. Fab fragments with different binding specificities dimerized with leucine zippers (e.g. c-fos and c-jun) replacing the constant domain; US Pat. Bispecific and oligospecific monovalent and oligovalent receptors, such as the VH-CH1 regions of two antibodies (two Fab fragments), as described, for example, in US Pat. A polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with an associated light chain; e.g. Bispecific DNA-antibody conjugates as described, e.g., cross-linking of antibodies or Fab fragments with double-stranded pieces of DNA; Multispecific fusion proteins, e.g., expression constructs containing two scFvs with a hydrophilic helical peptide linker between them and a complete constant region; polyvalent and multispecific binding proteins, e.g., polypeptides having a first domain comprising an Ig heavy chain variable region binding region and a second domain comprising an Ig light chain variable region binding region, commonly referred to as diabodies. Conformational structures that create dimers (bispecific, trispecific, or tetraspecific molecules of the peptide) are also disclosed; e.g., as described in U.S. Pat. No. 5,837,821. a minibody construct in which the linked VL and VH chains, which can dimerize to form a bispecific/multivalent molecule, are further linked to the antibody hinge region and CH3 region with a peptide spacer; VH and VL that are linked in either orientation with a short peptide linker (e.g., 5 or 10 amino acids) or without any linker that can merise to form a bispecific diabody. domains; trimers and tetramers as described e.g. in U.S. Pat. No. 5,844,094; at the C-terminus as e.g. Strings of VH domains (or VL domains within family members) associated with bridging groups by peptide bonds that further associate with VL domains to form a series of FVs (or scFvs); and U.S. Patent No. 5,869,620 A single chain binding polypeptide having both a VH domain and a VL domain linked by a peptide linker is combined into a multivalent structure by non-covalent or chemical cross-linking, e.g. , including those that form homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV-type or diabody-type formats. For additional exemplary multispecific and bispecific molecules and methods of making them, see, e.g., U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; , 959,083, U.S. Patent No. 5,989,830, U.S. Patent No. 6,005,079, U.S. Patent No. 6,239,259, U.S. Patent No. 6,294 , 353, U.S. Patent No. 6,333,396, U.S. Patent No. 6,476,198, U.S. Patent No. 6,511,663, U.S. Patent No. 6,670,453 US Patent No. 6,743,896, US Patent No. 6,809,185, US Patent No. 6,833,441, US Patent No. 7,129,330 U.S. Patent No. 7,183,076, U.S. Patent No. 7,521,056, U.S. Patent No. 7,527,787, U.S. Patent No. 7,534,866, U.S. Patent No. 7,612,181, U.S. Patent Application Publication No. 2002/004587A1, U.S. Patent Application Publication No. 2002/076406A1, U.S. Patent Application Publication No. 2002/103345A1, U.S. Pat. US Patent Application Publication No. 2003/207346A1, US Patent Application Publication No. 2003/211078A1, US Patent Application Publication No. 2004/219643A1, US Patent Application Publication No. 2004/220388A1, US Patent Application Publication No. No. 2004/242847A1, US Patent Application No. 2005/003403A1, US Patent Application No. 2005/004352A1, US Patent Application No. 2005/069552A1, US Patent Application No. 2005 /079170A1, US 2005/100543A1, US 2005/136049A1, US 2005/136051A1, US 2005/163782A1 specification, US Patent Application Publication No. 2005/266425A1, US Patent Application Publication No. 2006/083747A1, US Patent Application Publication No. 2006/120960A1, US Patent Application Publication No. 2006/204493A1 US Patent Application Publication No. 2006/263367A1, US Patent Application Publication No. 2007/004909A1, US Patent Application Publication No. 2007/087381A1, US Patent Application Publication No. 2007/128150A1, U.S. Patent Application Publication No. 2007/141049A1, U.S. Patent Application Publication No. 2007/154901A1, U.S. Patent Application Publication No. 2007/274985A1, U.S. Patent Application Publication No. 2008/050370A1, U.S. Patent Application No. US Patent Application Publication No. 2008/069820A1, US Patent Application Publication No. 2008/152645A1, US Patent Application Publication No. 2008/171855A1, US Patent Application Publication No. 2008/241884A1, US Patent Application Publication No. 2008/254512A1, US 2008/260738A1, US 2009/130106A1, US 2009/148905A1, US 2009 /155275A1, US Patent Application Publication No. 2009/162359A1, US Patent Application Publication No. 2009/162360A1, US Patent Application Publication No. 2009/175851A1, US Patent Application Publication No. 2009/175867A1 specification, US Patent Application Publication No. 2009/232811A1, US Patent Application Publication No. 2009/234105A1, US Patent Application Publication No. 2009/263392A1, US Patent Application Publication No. 2009/274649A1 WO 00/06605A2 pamphlet, WO 02/072635A2 pamphlet, WO 04/081051A1 pamphlet, WO 06/020258A2 pamphlet, WO 06/020258A2 pamphlet International Publication No. 2007/044887A2 pamphlet, International Publication No. 2007/095338A2 pamphlet, International Publication No. 2007/137760A2 pamphlet, International Publication No. 2008/119353A1 pamphlet, International Publication No. 2009/021754A2 pamphlet, International Publication No. 2009/068630A1 Pamphlet, International Publication No. 91/03493A1 pamphlet, International Publication No. 93/23537A1 pamphlet, International Publication No. 94/09131A1 pamphlet, International Publication No. 94/12625A2 pamphlet, International Publication No. 95/09917A1 pamphlet, International Publication No. Reference is made to pamphlet No. 96/37621A2 and pamphlet WO 99/64460A1.

"Fusion protein" and "fusion polypeptide" refer to a polypeptide having at least two moieties covalently linked together, each of those moieties having different properties. . The property can be a biological property, such as in vitro or in vivo activity. A property can also be a simple chemical or physical property, such as binding to a target molecule, catalyzing a reaction, etc. The two moieties may be directly linked by a single peptide bond or may be linked through a peptide linker but in reading frame with each other.

In certain embodiments, antibody molecules include diabodies and single chain molecules, as well as antigen-binding fragments of antibodies (eg, Fab, F(ab') ₂ , and Fv). For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH) and a light (L) chain variable domain sequence (abbreviated herein as VL). . In certain embodiments, the antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half-antibody). In another example, the antibody molecule comprises two heavy (H) chain variable domains. sequence and two light (L) chain variable domain sequences, thereby forming two antigen binding sites, e.g., Fab, Fab', F(ab') ₂ , Fc, Fd, Fd', Fv, These include single chain antibodies (e.g. scFv), single variable domain antibodies, diabodies (Dabs) (bivalent and bispecific), and chimeric (e.g. humanized) antibodies, which are made by modification of whole antibodies. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments are of any class of antibodies, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass of antibodies (e.g., IgG1, IgG2, IgG3, and IgG4). Preparations of antibody molecules may be monoclonal or polyclonal. Antibody molecules may also be human, humanized, CDR-grafted, or in vitro generated antibodies. An antibody may have a heavy chain constant region, e.g. IgG1, IgG2, IgG3, or IgG4. An antibody may also have a light chain, e.g. kappa or lambda. The term "immunoglobulin" (Ig) The term "antibody" is used interchangeably herein.

Examples of antigen-binding fragments of antibody molecules include (i) Fab fragments that are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) comprising two Fab fragments linked by a disulfide bridge in the hinge region. F(ab')2 fragment, which is a bivalent fragment; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VL and VH domains of a single arm of the antibody; (v) consisting of VH domain. diabody (dAb) fragments; (vi) camelid or camelid variable domains; (vii) single chain Fv (scFv), eg, as described by Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) single domain antibodies. 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 intact antibodies.

The term "antibody" includes intact molecules as well as functional fragments thereof. The constant region of an antibody is modified such that the properties of the antibody are modified (e.g., increased or can be altered, e.g. mutated, such that the

Antibody molecules may also be single domain antibodies. Single domain antibodies can include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from traditional four chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Can be mentioned. The single domain antibody may be any as described in the art or any future single domain antibody. Single domain antibodies can be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the invention, the single domain antibody is a naturally occurring single domain antibody known as a heavy chain antibody that lacks a light chain. Such single domain antibodies are disclosed, for example, in WO 94/04678 pamphlet. For clarity, this variable domain derived from a heavy chain antibody naturally lacking a light chain is referred to herein as a VHH or nanobody to distinguish it from the conventional VH of a four-chain immunoglobulin. known as. Such VHH molecules can be derived from antibodies raised in Camelidae species, such as camels, llamas, dromedaries, alpacas and guanacos. Other species other than Camelidae may also produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the present invention.

The VH and VL regions are subdivided into hypervariable regions called "complementarity-determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs or FWs). be able to.

The framework regions and CDR ranges have been precisely defined by a number of methods (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and AbM definitions used by Oxford Molecular's AbM antibody modeling software. About Generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Konterman, R., Springer-Verlag, Heidelberg).

The terms "complementarity determining region" and "CDR" as used herein refer to sequences of amino acids within antibody variable regions that confer antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3).

The exact amino acid sequence boundaries of a given CDR can be found in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al. , (1997) JMB 273, 927-948 (the "Chothia" numbering scheme). As used herein, CDRs defined by the "Chothia" numbering scheme are also sometimes referred to as "hypervariable loops."

For example, based on Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); The CDR amino acid residues of the variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Based on Chothia, the CDR amino acids of VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); the amino acid residues of VL are numbered 26-32 (LCDR1). ), 50-52 (LCDR2), and 91-96 (LCDR3). By combining both Kabat and Chothia CDR definitions, the CDRs include amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH, as well as amino acid residues 95-102 (HCDR3) of human VL. Consists of 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3).

In general, unless specifically indicated, an antibody molecule may include any combination of one or more Kabat CDRs and/or Chothia hypervariable loops. In one embodiment, the following definitions are used for the antibody molecules listed in Table 1: HCDR1 according to the combined CDR definitions of both Kabat and Chothia, and HCCDR2-3 and LCCDR1-3 according to the Kabat CDR definitions. In either definition, each VH and VL typically has three CDRs and four arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Including FR.

As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two, or more N-terminal or C-terminal amino acids, or include other changes compatible with the formation of protein structure. There is also.

The term "antigen binding site" refers to the part of an antibody molecule that contains determinants that form a junction that binds to a target (such as TGFβ) or an epitope thereof. For proteins (or protein mimetics), the antigen binding site typically includes one or more loops (of at least four amino acids or amino acid mimetics) that form a junction that binds to the target polypeptide. Typically, the antigen binding site of an antibody molecule comprises at least one or two CDRs and/or hypervariable loops, or more typically at least three, four, five or six CDRs and/or hypervariable loops. include.

The term "competes" or "cross-competes" as used herein means that an antibody molecule is targeted by another antibody molecule, e.g., an anti-TGFβ antibody molecule provided herein, e.g., TGFβ1, 2, or 3 used synonymously to refer to the ability to prevent binding to. Binding may be prevented directly or indirectly (eg, through allosteric modulation of the antibody molecule or target). The extent to which an antibody molecule is able to prevent binding to a target by another antibody molecule, and thus whether it can be said to compete, is determined using a competitive binding assay, e.g., a FACS assay, an ELISA or a BIACORE assay. be able to. In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, binding to the target by the first antibody molecule in a competitive binding assay (e.g., a competitive assay described herein) is 10% or more, such as 20% or more, 30% or more, 40% or more. % or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more When decreased, the first anti-TGFβ antibody molecule is said to compete with the second anti-TGFβ antibody molecule for binding to the target.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies may be made by hybridoma technology or by methods that do not involve hybridoma technology (eg, recombinant methods).

A "virtually human" protein is one that does not elicit a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. HAMA can be a problem under a number of circumstances, eg, when antibody molecules are administered repeatedly, eg, in the treatment of chronic or relapsing disease conditions. HAMA reactions increase antibody clearance from serum (see, e.g., Saleh et al., Cancer Immunol. Immunother. 32:180-190 (1990)), and also because of the potential for allergic reactions ( See, eg, LoBuglio et al., Hybridoma, 5:5117-5123 (1986)), repeated administration of antibodies can potentially be ineffective.

The antibody molecules described may be polyclonal or monoclonal antibodies. In other embodiments, antibodies may be made recombinantly, for example, by phage display or by combinatorial methods.

Phage display and combinatorial methods for making antibodies are known in the art (e.g., Ladner et al. US Pat. No. 5,223,409; Kang et al. WO 92/18619). Dower et al. International Publication No. 91/17271 pamphlet; Winter et al. International Publication No. 92/20791 pamphlet; Markland et al. International Publication No. 92/15679 pamphlet; Breitling et al. International Publication No. 93 /01288 pamphlet; McCafferty et al. WO 92/01047 pamphlet; Garrard et al. WO 92/09690 pamphlet; Ladner et al. WO 90/02 pamphlet 809; Fuchs et al. 1991) Bio/Technology 9:1370-1 372; Hay et al. (1992) Hum Antibody Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; ffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:35 76-3580 ; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88 :7978-7982 As per.

In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse that has been genetically engineered to produce the antibody from human immunoglobulin sequences) or a non-human antibody, e.g. or rat), goat, primate (eg, monkey), or camel antibodies. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods for producing rodent antibodies are known.

Human monoclonal antibodies can be produced using transgenic mice carrying human immunoglobulin genes rather than mouse systems. Splenocytes from these transgenic mice immunized with the antigen of interest are used to generate hybridomas that secrete human mAbs with specific affinity for epitopes from human proteins (e.g., Wood et al. Kucherlapati et al. WO 91/10741; Lonberg et al. WO 92/03918; Kay et al. WO 92/03917; Lonberg, N.; et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7: 13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81 :6851-6855;Bruggeman et al.1993 Year Immunol 7:33-40;Tuaillon et al.1993 PNAS 90:3720-3724;Bruggeman et al.1991 Eur J Immun ol 21:1323-1326).

The antibody may be one in which the variable region, or a portion thereof, eg, the CDRs, is produced in a non-human organism, eg, rat or mouse. Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the invention. Antibodies that are produced in a non-human organism, eg, a rat or a mouse, and then modified, eg, in the variable framework or constant regions, to reduce antigenicity in humans are within the scope of the invention.

Chimeric antibodies can be made by recombinant DNA techniques known in the art (Robinson et al., PCT/US86/02269; Akira, et al., European Patent No. 184,187). Taniguchi, M., European Patent No. 171,496; Morrison et al., European Patent No. 173,494; Neuberger et al., WO 86/01533; Cabilly et al. USA Patent No. 4,816,567; Cabilly et al., European Patent No. 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al., 1987, J. Immunol. 139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al., 1987, Canc. Res. 47: 999- 1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two, but generally all three recipient CDRs (of the heavy and/or light immunoglobulin chains) replaced with donor CDRs. The antibody may have at least a portion of the non-human CDRs replaced, or only a portion of the CDRs may be replaced by non-human CDRs. It is sufficient to replace as many CDRs as necessary for the humanized antibody to bind its target, eg TGFβ. Preferably, the donor will be a rodent antibody, such as a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin that provides the CDRs is called the "donor" and the immunoglobulin that provides the framework is called the "acceptor." In one embodiment, the donor immunoglobulin is non-human (eg, a rodent). The acceptor framework is a naturally occurring (eg, human) framework or a consensus framework, or a sequence that is about 85% or more, preferably 90%, 95%, 99% or more identical thereto.

As used herein, the term "consensus sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in a family of closely related sequences (e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a protein family, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in that family. Any may be included in a consensus sequence if they occur in frequency. "Consensus framework" refers to framework regions in a consensus immunoglobulin sequence.

Antibodies can be humanized by methods known in the art (e.g., Morrison, S.L., 1985, Science 229:1202-1207, Oi et al., 1986, BioTechniques 4:214, and Queen See U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761 and U.S. Pat. No. 5,693,762 by et al.

Humanized or CDR-grafted antibodies can be generated by CDR grafting or CDR replacement, in which one, two, or all CDRs of an immunoglobulin chain can be replaced. For example, US Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; See Winter US Pat. No. 5,225,539. Winter describes CDR grafting methods that can be used to prepare humanized antibodies of the invention (UK Patent Application No. 2,188,638A, filed March 26, 1987; Winter US Pat. No. 225,539).

Also within the scope of the invention are humanized antibodies in which specific amino acids have been substituted, deleted, or added. For selection criteria for amino acids from donors, see U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. It is described in columns 12 to 16 of the specification of No. 089. Other techniques for humanizing antibodies are described in Padlan et al., published December 23, 1992. It is described in European Patent Application No. 519596 A1.

The antibody molecule may be a single chain antibody. Single chain antibodies (scFVs) can be engineered (eg, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2: 245-52). Dimerizing or multimerizing single chain antibodies allows the creation of multivalent antibodies with specificity for different epitopes of the same target protein.

In still other embodiments, the antibody molecule comprises heavy chain constant regions, such as those of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, for example, IgG1, IgG2, It has IgG3 and IgG4 (human) heavy chain constant regions. In another embodiment, the antibody molecule has a light chain constant region, eg, a kappa or lambda (human) light chain constant region. Altering, e.g., mutating, the constant region modifies the properties of the antibody (e.g., changes in Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, and/or complement function). one or more of the above can be increased or decreased). In one embodiment, the antibody has effector functions; and is capable of fixing complement. In other embodiments, the antibody does not recruit effector cells; or fix complement. In another embodiment, the antibody has reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, eg it has mutagenesis or deletions in the Fc receptor binding region.

Methods of modifying antibody constant regions are known in the art. An antibody with altered function, e.g., an altered affinity for an effector ligand, such as an FcR on a cell, or the C1 component of complement, is an antibody in which at least one amino acid residue in the constant portion of the antibody is replaced by another residue. groups (see, e.g., EP-A-388,151 A1, US Pat. No. 5,624,821 and US Pat. thing). Similar modifications may be described that, if applied to immunoglobulins of mice, or other species, would reduce or eliminate their function.

An antibody molecule can be derivatized or linked to another functional molecule (eg, another peptide or protein). As used herein, a "derivatized" antibody molecule is one that has been modified. Derivatization methods include, but are not limited to, addition of affinity ligands such as fluorescent moieties, radionucleotides, toxins, enzymes or biotin. Accordingly, antibody molecules of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule may contain another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or an antibody or antibody portion and another molecule (such as a streptavidin core region or function (by chemical coupling, genetic fusion, non-covalent association or other methods) with one or more other molecular entities, such as proteins or peptides, that can mediate association with (such as a polyhistidine tag) can be linked together.

Certain derivatized antibody molecules are made by cross-linking two or more antibodies (of the same type or, eg, to create bispecific antibodies, of different types). Suitable crosslinkers include those that are heterobifunctional, with two differently reactive groups separated by a suitable spacer (e.g. m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional. Examples include those that are functional (eg, disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which the antibody molecules of the invention can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms, such as europium (Eu), and other anthanides, and radioactive materials (described below). Exemplary fluorescently detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies may also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, acetylcholinesterase, glucose oxidase. When an antibody is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, addition of hydrogen peroxide and diaminobenzidine leads to a detectable colored reaction product. Antibody molecules may also be derivatized with prosthetic groups such as streptavidin/biotin and avidin/biotin. For example, antibodies can be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; and bioluminescent substances. Examples of substances include luciferase, luciferin, and aequorin.

Labeled antibody molecules can be used, for example, for diagnostic and/or experimental purposes, by (i) isolating a given antigen by standard techniques such as affinity chromatography or immunoprecipitation; (ii) protein abundance; (iii) detecting a given antigen (e.g. in a cell lysate or cell supernatant) to evaluate the expression pattern and expression pattern; (iii) as part of a clinical trial procedure, e.g. to determine the effectiveness of a given treatment regimen; As such, it can be used in a number of contexts, including monitoring protein levels in tissues.

An antibody molecule can be conjugated to another molecular entity, typically a label or a therapeutic (eg, cytotoxic or cytostatic) agent or moiety. Radioactive isotopes can be used in diagnostic or therapeutic applications.

The present invention provides radiolabeled antibody molecules and methods for labeling the same. In one embodiment, a method of labeling antibody molecules is disclosed. The method involves contacting an antibody molecule with a chelating agent, thereby producing a conjugated antibody.

As discussed above, antibody molecules can be conjugated to therapeutic agents. The therapeutically active radioisotopes are as described above. Examples of other therapeutic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrasindione, mitox Santron, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, such as maytansinol (e.g., U.S. Pat. No. 5,208 ,020), CC-1065 (e.g., U.S. Pat. No. 5,475,092, U.S. Pat. No. 5,585,499, U.S. Pat. No. 5,846,545) ) and their analogs or homologs. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa ) Chlorambucil, CC-1065, Melphalan, Carmustine (BSNU) and Lomustine (CCNU), Cyclophosphamide, Busulfan, Dibromomannitol, Streptozotocin, Mitomycin C, and Cisdichlorodiamine Platinum(II) (DDP) Cisplatin ), anthracyclinies (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin (AMC)), Included are mitotic agents such as vincristine, vinblastine, taxol and maytansinoids.

In one aspect, the present disclosure provides methods of providing target binding molecules that specifically bind to targets disclosed throughout. For example, the target binding molecule is an antibody molecule. This method provides a method for detecting at least a portion of a non-human protein that is homologous (at least 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89) to a corresponding portion of a human target protein. at least one amino acid (e.g., at least 1, 2, 3, 4, 5, 6, 7, provide a target protein comprising moieties that differ by 8 or 9 amino acids); obtain antibody molecules that specifically bind to an antigen; and determine the effectiveness of the binding agent in modulating the activity of the target protein. Including evaluating. The method can further include administering a binding agent (eg, an antibody molecule) or a derivative (eg, a humanized antibody molecule) to a human subject.

This disclosure provides isolated nucleic acid molecules (ie, polynucleotides) encoding any of the antibody molecules described throughout. Also disclosed are vectors containing the nucleic acid molecules and host cells thereof. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

Combinations The therapeutic methods described herein may include two or more other therapeutic agents, procedures or modalities administered in combination.

In some embodiments, a TGF-β inhibitor (eg, NIS793) is administered in combination with a PD1 inhibitor (eg, an anti-PD1 antibody molecule). In some embodiments, the TGF-β inhibitor is administered on the same day as the PD1 inhibitor. In some embodiments, the TGF-β inhibitor is administered after administration of the PD1 inhibitor has begun. In some embodiments, the TGF-β inhibitor is administered 1 hour after administration of the PD1 inhibitor has ended.

In some embodiments, a TGFβ inhibitor (e.g., NIS793) is administered at a dose of 1300-1500 mg (e.g., about 1400 mg), e.g., once every two weeks, and a PD1 inhibitor (e.g., an anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 300 to 500 mg (eg, 400 mg), eg, once every four weeks.

In some embodiments, a TGFβ inhibitor (e.g., NIS793) is administered at a dose of 1300-1500 mg (e.g., about 1400 mg), e.g., once every two weeks, and a PD1 inhibitor (e.g., an anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every four weeks.

In some embodiments, a TGFβ inhibitor (e.g., NIS793) is administered at a dose of 1300-1500 mg (e.g., about 1400 mg), e.g., once every two weeks, and a PD1 inhibitor (e.g., an anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 1300-1500 mg (e.g., about 1400 mg), e.g., once every two weeks, and the PD1 inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 100 to 300 mg (eg, 200 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 1300-1500 mg (e.g., about 1400 mg), e.g., once every three weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 100 to 300 mg (eg, 200 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 1300-1500 mg (e.g., about 1400 mg), e.g., once every three weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every four weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every two weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 300 to 500 mg (eg, 400 mg), eg, once every four weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every two weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every four weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every two weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every two weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 100 to 300 mg (eg, 200 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every three weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 100 to 300 mg (eg, 200 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every three weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 300 to 500 mg (eg, 400 mg), eg, once every four weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every three weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every four weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every three weeks, and the TGFβ inhibitor (e.g., anti-PD1 antibody molecule eg, spartalizumab or tislelizumab) is administered at a dose of 200 to 400 mg (eg, 300 mg), eg, once every three weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., once every four weeks (e.g., on day 1 of a 28-day cycle). , and the PD1 inhibitor (eg, anti-PD1 antibody molecule, eg, spartalizumab or tislelizumab) is administered at a dose of 200-400 mg (eg, 300 mg), eg, once every four weeks.

In some embodiments, the TGFβ inhibitor (e.g., NIS793) is administered at a dose of 2000-2200 mg (e.g., about 2100 mg), e.g., twice every four weeks (e.g., on days 1 and 15 of a 28-day cycle). ) and the PD1 inhibitor (eg, anti-PD1 antibody molecule, eg, spartalizumab or tislelizumab) is administered at a dose of 200-400 mg (eg, 300 mg), eg, once every four weeks.

In certain embodiments, the methods described herein include antibody molecules, chemotherapeutic agents, other anti-cancer therapies (e.g., targeted anti-cancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapies, or oncolytics), cytotoxic agents, immune-based therapies (e.g., cytokine- or cell-based immunotherapy), surgical procedures (e.g., lumpectomy or mastectomy), or radiological procedures, or the foregoing. may be administered with one or more other therapeutic agents, including combinations of any one of the following. Additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is an enzyme inhibitor (eg, a small molecule enzyme inhibitor) or a metastasis inhibitor. Exemplary cytotoxic agents that may be administered in combination include anti-microtubule agents, topoisomerase inhibitors, antimetabolites, antimitotic agents, alkylating agents, anthracyclines, vinca alkaloids, intercalates. agents, agents capable of interfering with signal transduction pathways, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., local or whole body radiation (e.g., gamma irradiation). In other embodiments, The additional therapy is surgery or radiation, or a combination thereof. In other embodiments, the additional therapy is a therapy that targets one or more of the PI3K/AKT/mTOR pathway, an HSP90 inhibitor. , or a tubulin inhibitor.

Alternatively, or in combination with the foregoing, the methods described herein can be used with immunomodulatory agents (e.g., activators of costimulatory molecules or inhibitory molecules, e.g., inhibitors of immune checkpoint molecules); vaccines, e.g. , therapeutic cancer vaccines; or other forms of cellular immunotherapy.

In certain embodiments, the combinations described herein are administered or used with co-stimulatory or inhibitory molecules, such as co-inhibitory ligands or receptor modulators.

In one embodiment, the combinations described herein are administered or used in combination with inhibitors of the suppressive (or immune checkpoint) molecules PD-1, PD-L1, PD-L2, and/or TGFβ. Ru. In one embodiment, the inhibitor is an antibody or antibody fragment that binds PD-1, PD-L1, PD-L2, or TGFβ.

For combination therapy, in some embodiments, the TGF-β inhibitor is administered on the same day as the checkpoint inhibitor. In other embodiments, the TGF-β inhibitor is administered before checkpoint inhibitor administration is complete. In further embodiments, the TGF-β inhibitor is administered after administration of the checkpoint inhibitor is completed. In some embodiments, the TGF-β inhibitor is administered at the same time as the checkpoint inhibitor. In some embodiments, the TGF-β inhibitor is given until remission (partial or complete) is achieved. In some embodiments, checkpoint inhibitors are given until remission (partial or complete) is achieved.

Compounds of the present disclosure can be administered in therapeutically effective amounts in combination therapy with one or more therapeutic agents (pharmaceutical combinations) or modalities, such as non-drug therapies. For example, synergistic effects with other anti-cancer agents may occur. When a compound of the present application is administered in conjunction with other treatments, the dosage of the co-administered compound will, of course, depend on the type of concomitant drug used, the specific drug used, the disease state being treated, etc. It will be different.

The compounds can be administered simultaneously (in a single formulation or in separate formulations), sequentially, separately, or over a period of time with other drug therapies or treatment modalities. Generally, combination therapy contemplates the administration of two or more drugs during a single cycle or course of treatment. A therapeutic agent is, for example, a chemical compound, peptide, antibody, antibody fragment, or nucleic acid that has or enhances therapeutic activity when administered to a patient in combination with a compound of the present disclosure.

In one aspect, the TGFβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) is a drug that is used in combination with other anticancer agents, antiallergic agents, antiemetics (or antiemetics), analgesics, cytoprotective agents, and It can be used in combination with other therapeutic agents, such as combinations of these.

In some embodiments, the TGFβ inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a LAG-3 inhibitor, a cytokine, an A2A antagonist, a GITR agonist, a TIM inhibitor, for the treatment of a disease, such as cancer. -3 inhibitors, STING agonists, and TLR7 agonists.

In another embodiment, one or more chemotherapeutic agents are used in combination with a TGFβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) for the treatment of a disease, such as cancer, wherein The chemotherapeutic agents include, but are not limited to, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), and busulfan (Myleran®). )), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU) ® ), chlorambucil (Leukeran ® ), cisplatin (Platinol ® ), cladribine (Leustatin ® ), cyclophosphamide (Cytoxan ® or Neosar ® ) , cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan) , daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®) ), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycytidine), Hydroxyurea (Hydrea®), Idarubicin (Idamycin®), Ifosfamide (IFEX®), Irinotecan (Camptosar®), L-Asparaginase (ELSPAR®) ), leucovorin calcium, melphalan (Alkeran(R)), 6-mercaptopurine (Purinethol(R)), methotrexate (Folex(R)), mitoxantrone (Novantrone(R)), Mylotarg, paclitaxel (Taxol®) or nab-paclitaxel, Phoenix (Yttrium 90/MX-DTPA), pentostatin, polyfeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex) (R)), teniposide (Vumon(R)), 6-thioguanine, thiotepa, tirapazamine (Tirazone(R)), topotecan hydrochloride for injection (Hycamptin(R)), vinblastine (Velban(R)) , vincristine (Oncovin®), vinorelbine (Navelbine®), epirubicin (Elence®), oxaliplatin (Eloxatin®), exemestane (Aromasin®), letrozole ( Femara®) and fulvestrant (Faslodex®). For example, a TGFβ inhibitor (eg, NIS793) can be used in combination with gemcitabine. In another example, a TGFβ inhibitor (eg, NIS793) can be used in combination with nab-paclitaxel. TGFβ inhibitors (eg, NIS793) can be used in combination with both gemcitabine and nab-paclitaxel. In a further example, a TGFβ inhibitor (eg, NIS793) can be used in combination with cyclophosphamide. TGFβ inhibitors (eg, NIS793) may also be used in combination with topotecan. In some examples, a TGFβ inhibitor (eg, NIS793) can be used in combination with both cyclophosphamide or topotecan. In some examples, a TGFβ inhibitor (eg, NIS793) may be combined with 5-fluorouracil, leucovorin (or levoleucovorin), irinotecan, bevacizumab, and optionally tislelizumab. In some examples, a TGFβ inhibitor (eg, NIS793) may be combined with 5-fluorouracil, leucovorin (or levoleucovorin), oxaliplatin, bevacizumab, and optionally tislelizumab. In some instances, a TGFβ inhibitor (eg, NIS793) may be used in combination with oxaliplatin and capecitabine. In some examples, a TGFβ inhibitor (eg, NIS793) may be used in combination with oxaliplatin, leucovorin (or levoleucovorin), and 5-fluorouracil.

In other embodiments, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure include one or more other anti-HER2 antibodies described above, such as trastuzumab, Used in combination with pertuzumab, margetuximab or HT-19, or other anti-HER2 conjugates such as ado-trastuzumab emtansine (also known as Kadcyla®, or T-DM1).

In other embodiments, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are EGFR inhibitors, Her3 inhibitors, IGFR inhibitors, for the treatment of diseases, such as cancer. used in combination with one or more tyrosine kinase inhibitors, including, but not limited to, tyrosine kinase inhibitors and Met inhibitors.

For example, tyrosine kinase inhibitors include erlotinib hydrochloride (Tarceva®); linifanib (N-[4-(3-amino-1H-indazole- Sunitinib malate (Sutent®); Bosutinib (also known as SKI-606, US Pat. No. 6,780) , 996, (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline -3-carbonitrile); dasatinib (Sprycel®); pazopanib (Votrient®); sorafenib (Nexavar®); Zactima (ZD6474); and imatinib or imatinib mesylate (Gilvec®) ) and Gleevec®).

Epidermal growth factor receptor (EGFR) inhibitors include erlotinib hydrochloride (Tarceva®), gefitinib (Iressa®); N-[4-[(3-chloro-4-fluorophenyl)amino ]-7-[[(3''S'')-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide, Tovok®); vandetanib (Caprelsa ( Lapatinib (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1, 2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514); canertinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl] -N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-0); mubritinib (TAK165); peritinib (EKB569); Afatinib (Gilotrif®); Neratinib (HKI-272); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[ 2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester (BMS599626); N-(3,4-dichloro-2-fluoro phenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolineamine (XL647, CAS 781613-23- 8); and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS 187724-61-4 ), but are not limited to these.

EGFR antibodies include cetuximab (Erbitux®); panitumumab (Vectibix®); matuzumab (EMD-72000); nimotuzumab (hR3); zaltumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1 ); and ch806 (mAb-806, CAS 946414-09-1).

Other HER2 inhibitors include neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano- 7-ethoxyquinolin-6-yl]-4-(dimethylamino)buter-2-enamide, described in PCT Publication No. WO 05/028443); lapatinib or lapatinib tosylate (Tykerb®) , (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidine -3-ol (BMS690514); (2E)-N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6- quinazolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6); N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazole-5 -yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinyl methyl ester (BMS 599626, CAS 714971 -09-2); canertinib dihydrochloride (PD183805 or CI-1033); and N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)- Examples include, but are not limited to, octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8).

Her3 inhibitors include, but are not limited to, LJM716, MM-121, AMG-888, RG7116, REGN-1400, AV-203, MP-RM-1, MM-111 and MEHD-7945A.

MET inhibitors include cabozantinib (XL184, CAS 849217-68-1); foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); tivantinib (ARQ197, CAS 1000873-98-2); 1-(2 -Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H -Pyrazole-4-carboxamide (AMG 458); crizotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-dihydro-1H-indol-1-ylsulfonyl)-3-( {3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indol-2-one (SU11271) ;(3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)- N-Methyl-2-oxoindoline-5-sulfonamide (SU11274); (3Z)-N-(3-chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin-4-ylpropyl) )-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide (SU11606); 6-[difluoro[6-(1-methyl-1H-pyrazol-4-yl)] -1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]-quinoline (JNJ38877605, CAS 943540-75-8); 2-[4-[1-(quinolin-6-ylmethyl) )-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl]-1H-pyrazol-1-yl]ethanol (PF04217903, CAS 956905-27-4); N-(( 2R)-1,4-dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5] Cyclohepta[1,2-b]pyridin-7-yl]sulfamide (MK2461, CAS 917879-39-1); 6-[[6-(1-methyl-1H-pyrazol-4-yl)-1,2, 4-triazolo[4,3-b]pyridazin-3-yl]thio]-quinoline (SGX523, CAS 1022150-57-7); and (3Z)-5-[[(2,6-dichlorophenyl)methyl]sulfonyl ]-3-[[3,5-dimethyl-4-[[(2R)-2-(1-pyrrolidinylmethyl)-1-pyrrolidinyl]carbonyl]-1H-pyrrol-2-yl]methylene]-1 ,3-dihydro-2H-indol-2-one (PHA665752, CAS 477575-56-7).

IGFR inhibitors include, but are not limited to, BMS-754807, XL-228, OSI-906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573 and BI836845 . For an overview, see, eg, Yee, JNCI, 104; 975 (2012).

In another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are inhibitors, BRAF inhibitors, PI3K/ Used in combination with one or more proliferation signaling pathway inhibitors, including, but not limited to, Akt inhibitors, SHP2 inhibitors, and also mTOR inhibitors and CDK inhibitors.

For example, mitogen-activated protein kinase (MEK) inhibitors include XL-518 (also known as GDC-0973, available from CAS No. 1029872-29-4, ACC Corp.); 2-[(2- chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (also known as CI-1040 or PD184352, described in PCT Application No. WO 2000035436) ;N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (also known as PD0325901, PCT Application No. International 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butane dinitrile (also known as U0126, described in U.S. Pat. No. 2,779,780); N-[3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3-dihydroxy (3S,4R,5Z,8S,9S,11E)-14-(ethylamino )-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] (also known as E6201) 2'-amino-3'-methoxyflavone (also known as PD98059, available from Biaffin GmbH & Co., KG, Germany); -3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H) -dione (TAK-733, CAS 1035555-63-5); pimasertib (AS-703026, CAS 1204531-26-9); and trametinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80). , but not limited to.

BRAF inhibitors include vemurafenib (or Zelboraf®, PLX-4032, CAS 918504-65-1), GDC-0879, PLX-4720 (available from Symansis), dabrafenib (or GSK2118436), LGX 818, Examples include, but are not limited to, CEP-32496, UI-152, RAF 265, regorafenib (BAY 73-4506), CCT239065, or sorafenib (or sorafenib tosylate or Nexavar®).

As a phosphoinositide 3-kinase (PI3K) inhibitor, 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2 -d]pyrimidin-4-yl]morpholine (also known as GDC0941, RG7321, GNE0941, pictrellisib, or pictilisib, described in PCT publication numbers WO 09/036082 and WO 09/055730) ); tozasertib (VX680 or MK-0457, CAS 639089-54-6), (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6 ,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10( 1H)-trione (PX866, CAS 502632-66-8); 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6), (S)-N1 -(4-Methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide (also known as BYL719 or alpelisib); 2-(4-(2-(1-isopropyl-3-methyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f ] imidazo[1,2-d][1,4]oxazepin-9-yl)-1H-pyrazol-1-yl)-2-methylpropanamide (also known as GDC0032, RG7604, or taselisib). However, it is not limited to these.

mToR inhibitors include temsirolimus (Torisel®); ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R) , 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl- 2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl] propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669 and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin ( AY22989, Sirolimus®); simapimod (CAS 164301-51-3); (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d] pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055), 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4 -methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N ² -[1,4-dioxo-4-[[4-(4- Oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L- -aspartyl L-serine, inner salt (SF1126, CAS 936487- 67-1), but are not limited to these.

CDK inhibitors include palbociclib (also known as PD-0332991, Ibrance®, 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl] amino}pyrido[2,3-d]pyrimidin-7(8H)-one).

In yet another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are IAP inhibitors, BCL2 inhibitors, Used in combination with one or more pro-apoptotic agents including, but not limited to, MCl1 inhibitors, TRAIL agents, CHK inhibitors.

For example, IAP inhibitors include, but are not limited to, LCL161, GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include WO 04/005284 pamphlet, WO 04/007529 pamphlet, WO 05/097791 pamphlet, WO 05/069894 pamphlet, WO 05 /069888 pamphlet, International Publication No. 05/094818 pamphlet, US Patent Application Publication No. 2006/0014700 specification, US Patent Application Publication No. 2006/0025347 specification, International Publication No. 06/069063 pamphlet, International Publication No. Examples include, but are not limited to, those disclosed in WO 06/010118 pamphlet, WO 06/017295 pamphlet, and WO 08/134679 pamphlet.

As a BCL-2 inhibitor, 4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4 -[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (also known as ABT-263) Tetrocalcin A; Antimycin; Gossypol ((-)BL-193); Obatoclax; Ethyl-2-amino-6-cyclopentyl- 4-(1-cyano-2-ethoxy-2-oxoethyl)-4H chromone-3-carboxylate (HA14-1); oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-gossypol acetic acid (AT-101); 4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)- 3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9); and navitoclax (ABT-263) , CAS 923564-51-6).

Pro-apoptotic receptor agonists (PARA) including DR4 (TRAILR1) and DR5 (TRAILR2) include duranermin (AMG-951, RhApo2L/TRAIL); mapatumumab (HRS-ETR1, CAS 658052-09-6); lexatumumab ( HGS-ETR2, CAS 845816-02-6); Apomab®; conatumumab (AMG655, CAS 896731-82-1); and tigatuzumab (CS1008, CAS 946415-34-5, available from Daiichi Sankyo) ), but are not limited to these.

Checkpoint kinase (CHK) inhibitors include 7-hydroxystaurosporine (UCN-01); 6-bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3 -piperidinylpyrazolo[1,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-6); 5-(3-fluorophenyl)-3-ureidothiophene-2-carboxylic acid N-[ (S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1-azabicyclo[2.2.2]oct-3-yl)amino]-3 -(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR124, CAS 405168-58-3); 7-aminodactinomycin (7-AAD), isogranulati Mido, debromohymenialdisine; N-[5-bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]-phenyl]-N'-(5-methyl-2-pyrazinyl) urea (LY2603618, CAS 911222-45-2); Sulforaphane (CAS 4478-93-7, 4-methylsulfinylbutyl isothiocyanate); 9,10,11,12-tetrahydro-9,12-epoxy-1H-diindolo[ 1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-1,3(2H)-dione (SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRQRRRLYRSPAMPENL (SEQ ID NO: 318)) and CBP501 ((d-Bpa)sws(d-Phe-F5)(d-Cha)rrrqrr), but are limited to these Not done.

In further embodiments, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used as one or more immunomodulators (e.g., for the treatment of a disease, e.g., cancer). activators of costimulatory molecules or inhibitors of immune checkpoint molecules).

In certain embodiments, the immunomodulator is an activator of costimulatory molecules. In one embodiment, the costimulatory molecule agonist is OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40. , BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or a CD83 ligand (eg, an agonist antibody or antigen-binding fragment thereof, or a soluble fusion).

GITR Agonists In some embodiments, GITR agonists are used in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) for the treatment of diseases, such as cancer. In some embodiments, the GITR agonist is GWN323 (NVS), BMS-986156, MK-4166 or MK-1248 (Merck), TRX518 (Leap Therapeutics), INCAGN1876 (Incyte/Agenus), AMG228 (Amg en) or INBR -110 (Inhibrx).

Exemplary GITR Agonists In one embodiment, the GITR agonist is an anti-GITR antibody molecule. In one embodiment, the GITR agonist is disclosed in International Publication No. 2016/057 entitled "Compositions and Methods of Use for Augmented Immune Response and Cancer Therapy" published on April 14, 2016. Anti-GITR described in pamphlet No. 846 It is an antibody molecule.

In one embodiment, the anti-GITR antibody molecule is shown in Table 5 (e.g., from the MAB7 heavy and light chain variable region sequences disclosed in Table 5) or is encoded by the nucleotide sequence shown in Table 5. at least 1, 2, 3, 4, 5, or 6 complementarity determining regions (CDRs) (or all CDRs taken together) from heavy and light chain variable regions comprising amino acid sequences comprising the following: In some embodiments, the CDRs follow the Kabat definition. In some embodiments, the CDRs follow the Chothia definition. In one embodiment, one or more of the CDRs (or all CDRs collectively) have 1, 2, 3, 4, 5, 6 or more changes, e.g., encoded by the nucleotide sequence or amino acid sequence (e.g., conservative amino acid substitutions) or deletions.

In one embodiment, the anti-GITR antibody molecule comprises a heavy chain variable region (VH ); and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 114, the VLCDR2 amino acid sequence of SEQ ID NO: 116, and the VLCDR3 amino acid sequence of SEQ ID NO: 118.

In one embodiment, the anti-GITR antibody molecule comprises an amino acid sequence of SEQ ID NO: 101, or a VH comprising an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 101. In one embodiment, the anti-GITR antibody molecule comprises the amino acid sequence of SEQ ID NO: 102, or a VL that includes an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 102. In one embodiment, the anti-GITR antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 101 and a VL comprising the amino acid sequence of SEQ ID NO: 102.

In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 105, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 105. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 106, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 106. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 105 and a VL encoded by the nucleotide sequence of SEQ ID NO: 106.

In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 103. In one embodiment, the anti-GITR antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 104. In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 104.

In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 107, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 107. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 108, or a nucleotide sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 108. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 107 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 108.

The antibody molecules described herein can be produced by the vectors, host cells and methods described in WO 2016/057846.

Other Exemplary GITR Agonists In one embodiment, the anti-GITR antibody molecule is BMS-986156 (Bristol-Myers Squibb), also known as BMS 986156 or BMS986156. BMS-986156 and other anti-GITR antibodies are disclosed, for example, in US Pat. No. 9,228,016 and WO 2016/196792. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of BMS-986156, e.g., as disclosed in Table 6, the heavy chain or the light chain variable region. sequence, or heavy chain or light chain sequence.

In one embodiment, the anti-GITR antibody molecule is MK-4166 or MK-1248 (Merck). MK-4166, MK-1248 and other anti-GITR antibodies are described, for example, in US Pat. No. 8,709,424, WO 2011/028683, WO 2015/026684 and Mahne et al. ．． Cancer Res. 2017;77(5):1108-1118. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences of MK-4166 or MK-1248 (or all CDR sequences taken together), the heavy or light chain variable region sequences, or the heavy or light chain variable region sequences of MK-4166 or MK-1248. Contains light chain sequences.

In one embodiment, the anti-GITR antibody molecule is TRX518 (Leap Therapeutics). TRX518 and other anti-GITR antibodies are described, for example, in US Pat. No. 7,812,135, US Pat. No. 8,388,967, US Pat. No. 9,028,823, and International Publication No. 2006/105021 pamphlet and Ponte J et al. (2010) Clinical Immunology; 135:S96. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences (or all CDR sequences collectively), a heavy or light chain variable region sequence, or a heavy or light chain sequence of TRX518.

In one embodiment, the anti-GITR antibody molecule is INCAGN1876 (Incyte/Agenus). INCAGN1876 and other anti-GITR antibodies are disclosed, for example, in US Patent Application Publication No. 2015/0368349 and WO 2015/184099. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences of INCAGN1876 (or all CDR sequences collectively), a heavy or light chain variable region sequence, or a heavy or light chain sequence.

In one embodiment, the anti-GITR antibody molecule is AMG 228 (Amgen). AMG 228 and other anti-GITR antibodies are disclosed, for example, in US Pat. No. 9,464,139 and WO 2015/031667. In one embodiment, the anti-GITR antibody molecule comprises one or more of the CDR sequences of AMG 228 (or all CDR sequences collectively), a heavy or light chain variable region sequence, or a heavy or light chain sequence. .

In one embodiment, the anti-GITR antibody molecule is INBRX-110 (Inhibrx). INBRX-110 and other anti-GITR antibodies are disclosed, for example, in US Patent Application Publication No. 2017/0022284 and WO 2017/015623. In one embodiment, the GITR agonist comprises one or more of the CDR sequences (or all CDR sequences collectively), a heavy or light chain variable region sequence, or a heavy or light chain sequence of INBRX-110.

In one embodiment, the GITR agonist (eg, fusion protein) is MEDI 1873 (MedImmune), also known as MEDI1873. MEDI 1873 and other GITR agonists are described, for example, in US Patent Application Publication No. 2017/0073386, WO 2017/025610 and Ross et al. Disclosed in Cancer Res 2016; 76 (14 Suppl): Abstract nr 561. In one embodiment, the GITR agonist comprises one or more of an IgG Fc domain, a functional multimerization domain, and a receptor binding domain of MEDI 1873 glucocorticoid-inducible TNF receptor ligand (GITRL).

Additional known GITR agonists (eg, anti-GITR antibodies) include, for example, those described in WO 2016/054638.

In one embodiment, the anti-GITR antibody is an antibody that competes for binding to and/or binds to the same epitope on GITR as one of the anti-GITR antibodies described herein.

In one embodiment, the GITR agonist is a peptide that activates the GITR signaling pathway. In one embodiment, the GITR agonist is an immunoadhesin-binding fragment (e.g., an immunoadhesin-binding fragment comprising the extracellular portion of GITRL or the GITR-binding portion) fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). be.

In certain embodiments, the immunomodulator is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulator is an inhibitor of PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFRβ. In one embodiment, the inhibitor of immune checkpoint molecules inhibits PD-1, PD-L1, LAG-3, TIM-3 or CTLA4, or any combination thereof.

Inhibition of inhibitory molecules can be performed at the DNA, RNA or protein level. In some embodiments, inhibitory nucleic acids (eg, dsRNA, siRNA or shRNA) can be used to inhibit expression of the inhibitory molecule. In other embodiments, the inhibitor of the inhibitory signal is a polypeptide, e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig), or an antibody or antigen-binding fragment thereof that binds to an inhibitory molecule; e.g. Antibodies or fragments thereof that bind to PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFRβ, or combinations thereof (also called “antibody molecules”).

In one embodiment, the antibody molecule is a complete antibody or a fragment thereof (eg, a Fab, F(ab')2, Fv, or single chain Fv fragment (scFv)). In yet other embodiments, the antibody molecule is selected from the heavy chain constant regions of, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; particularly, for example, IgG1, IgG2, IgG3 and It has a heavy chain constant region (Fc) selected from the heavy chain constant regions of IgG4, more particularly IgG1 or IgG4 (eg, human IgG1 or IgG4). In one embodiment, the heavy chain constant region is human IgG1 or human IgG4. In one embodiment, the constant region modifies a property of the antibody molecule (e.g., increases one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function or modified, e.g., mutated, so as to reduce

In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for PD-1 or PD-L1 and a second binding specificity for, for example, TGFβ, TIM-3, LAG-3, or PD-L1. It has a second binding specificity for L2. In one embodiment, the bispecific antibody molecule binds PD-1 or PD-L1 and TIM-3. In another embodiment, the bispecific antibody molecule binds PD-1 or PD-L1 and TGFβ. In another embodiment, the bispecific antibody molecule binds PD-1 and TGFβ. Any combination of the above molecules may be a multispecific antibody molecule, e.g., a first binding specificity for PD-1 or PD-1 and two of TGFβ, TIM-3, LAG-3, or PD-L2. Trispecific antibodies can be made that include second and third binding specificities for more than one.

In certain embodiments, the immunomodulator is an inhibitor of PD-1, eg, human PD-1. In another embodiment, the immunomodulator is an inhibitor of PD-L1, eg, human PD-L1. In one embodiment, the inhibitor of PD-1 or PD-L1 is an antibody molecule directed against PD-1 or PD-L1. PD-1 or PD-L1 inhibitors can be administered alone or in combination with other immunomodulators, eg, in combination with inhibitors of TGFβ, LAG-3, TIM-3 or CTLA4. In an exemplary embodiment, an inhibitor of PD-1 or PD-L1, e.g., an anti-TGFβ, anti-PD-1 or PD-L1 antibody molecule, is combined with a LAG-3 inhibitor, e.g., an anti-LAG-3 antibody molecule. Administered in combination. In another embodiment, an inhibitor of TGFβ, PD-1 or PD-L1, e.g., an anti-TGFβ, anti-PD-1 or PD-L1 antibody molecule, is a TIM-3 inhibitor, e.g., an anti-TIM-3 antibody molecule. Administered in combination with In still other embodiments, inhibitors of TGFβ, PD-1 or PD-L1, e.g., anti-TGFβ or anti-PD-1 antibody molecules, are inhibitors of LAG-3, e.g., anti-LAG-3 antibody molecules and TIM- 3 inhibitor, eg, an anti-TIM-3 antibody molecule.

Other combinations of PD-1 inhibitors and immunomodulators (e.g., one or more of PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFR) Within the scope of this disclosure. Any of the antibody molecules known in the art or disclosed herein can be used in the above combinations of inhibitors of checkpoint molecules.

CTLA-4 Inhibitors In some embodiments, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used to inhibit CTLA-4 for the treatment of a disease, such as cancer. Used in combination with drugs. In some embodiments, the PD-1 inhibitor is ipilimumab (MDX-010, MDX-101, or Yervoy, Bristol-Myers Squibb), tremelimumab (ticilimumab, Pfizer/AstraZeneca), AGEN1181 ( Agenus), selected from zarifrelimab (AGEN1884, Agenus), IBI310 (Innovent Biologics),

LAG-3 Inhibitors In some embodiments, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used to inhibit LAG-3 for the treatment of a disease, such as cancer. Used in combination with drugs. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), or TSR-033 (Tesaro).

Exemplary LAG-3 Inhibitors In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitors are those disclosed in U.S. Patent Application Publication No. 2015/0259420, published September 17, 2015, entitled "Antibody Molecules to LAG-3 and Uses Thereof." This is an anti-LAG-3 antibody molecule.

In one embodiment, the anti-LAG-3 antibody molecule is shown in Table 7 (e.g., from the heavy and light chain variable region sequences of BAP050-Clone I or BAP050-Clone J disclosed in Table 7), or At least 1, 2, 3, 4, 5, or 6 complementarity determining regions (CDRs) (or summarized Contains all CDRs). In some embodiments, the CDRs follow the Kabat definition (eg, as described in Table 7). In some embodiments, the CDRs follow the Chothia definition (eg, as described in Table 7). In some embodiments, the CDRs follow the combined CDR definitions of both Kabat and Chothia (eg, as described in Table 7). In one embodiment, the combination of Kabat and Chothia CDRs of VH CDR1 comprises the amino acid sequence GFTLTNYGMN (SEQ ID NO: 122). In one embodiment, one or more of the CDRs (or all CDRs collectively) exhibit 1, 2, 3, 4, 5, 6 or more changes, e.g., in Table 7; have amino acid substitutions (eg, conservative amino acid substitutions) or deletions to the amino acid sequence encoded by the nucleotide sequence shown in .

In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain variable region comprising a VHCDR1 amino acid sequence of SEQ ID NO: 123, a VHCDR2 amino acid sequence of SEQ ID NO: 124, and a VHCDR3 amino acid sequence of SEQ ID NO: 125, each disclosed in Table 7. (VH); and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 132, the VLCDR2 amino acid sequence of SEQ ID NO: 133, and the VLCDR3 amino acid sequence of SEQ ID NO: 134.

In one embodiment, the anti-LAG-3 antibody molecule comprises VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 158 or 159, VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 160 or 161, and the sequences disclosed in Table 7, respectively. VH comprising VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 162 or 163; and VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 168 or 169, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 170 or 171 and SEQ ID NO: 172 or 173 VL containing VLCDR3 encoded by the nucleotide sequence of. In one embodiment, the anti-LAG-3 antibody molecule comprises VHCDR1 encoded by the nucleotide sequence of SEQ ID NO: 180 or 159, VHCDR2 encoded by the nucleotide sequence of SEQ ID NO: 181 or 161, and the sequences disclosed in Table 7, respectively. VH comprising VHCDR3 encoded by the nucleotide sequence of SEQ ID NO: 182 or 163; and VLCDR1 encoded by the nucleotide sequence of SEQ ID NO: 168 or 169, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO: 170 or 171 and SEQ ID NO: 172 or 173 VL containing VLCDR3 encoded by the nucleotide sequence of.

In one embodiment, the anti-LAG-3 antibody molecule comprises a VH that comprises the amino acid sequence of SEQ ID NO: 128, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 128. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 140. In one embodiment, the anti-LAG-3 antibody molecule comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 146. including. In one embodiment, the anti-LAG-3 antibody molecule comprises the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 152. including. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 128 and a VL comprising the amino acid sequence of SEQ ID NO: 140. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 146 and a VL comprising the amino acid sequence of SEQ ID NO: 152.

In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 129 or 130, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 129 or 130. Includes VH. In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 141 or 142, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 141 or 142. Includes VL. In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 147 or 148, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 147 or 148. Includes VH. In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 153 or 154, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 153 or 154. Includes VL. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 129 or 130 and a VL encoded by the nucleotide sequence of SEQ ID NO: 141 or 142. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 147 or 148 and a VL encoded by the nucleotide sequence of SEQ ID NO: 153 or 154.

In one embodiment, the anti-LAG-3 antibody molecule comprises the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 131. Including chains. In one embodiment, the anti-LAG-3 antibody molecule comprises an amino acid sequence of SEQ ID NO: 143, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 143. Including chains. In one embodiment, the anti-LAG-3 antibody molecule comprises the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 149. Including chains. In one embodiment, the anti-LAG-3 antibody molecule comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 155. Including chains. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 131 and a light chain comprising the amino acid sequence of SEQ ID NO: 143. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 149 and a light chain comprising the amino acid sequence of SEQ ID NO: 155.

In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 138 or 139, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 138 or 139. Contains heavy chains. In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 144 or 145, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 144 or 145. Contains light chains. In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 150 or 151, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 150 or 151. Contains heavy chains. In one embodiment, the antibody molecule is encoded by a nucleotide sequence of SEQ ID NO: 156 or 157, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 156 or 157. Contains light chains. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 138 or 139 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 144 or 145. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 150 or 151 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 156 or 157.

The antibody molecules described herein may be made by vectors, host cells, and methods described in US Patent Application Publication No. 2015/0259420.

Other Exemplary LAG-3 Inhibitors In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986016 (Bristol-Myers Squibb), also known as BMS986016. BMS-986016 and other anti-LAG-3 antibodies are disclosed in WO 2015/116539 and US Patent No. 9,505,839. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences of BMS-986016 (or all CDR sequences taken together), e.g., as disclosed in Table 8, the heavy chain or the light chain. including variable region sequences, or heavy or light chain sequences.

In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tesaro). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences of TSR-033 (or all CDR sequences taken together), the heavy chain or light chain variable region sequence, or the heavy or light chain Contains arrays.

In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima BioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO 2008/132601 and US Patent No. 9,244,059. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of IMP731, e.g., the heavy chain or light chain variable region as disclosed in Table 8. sequence, or heavy chain or light chain sequence. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences (or all CDR sequences collectively), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence of GSK2831781. include.

In one embodiment, the anti-LAG-3 antibody molecule is IMP761 (Prima BioMed). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the CDR sequences of IMP761 (or all CDR sequences collectively), the heavy chain or light chain variable region sequence, or the heavy chain or light chain sequence. include.

Further known anti-LAG-3 antibodies include, for example, WO 2008/132601 pamphlet, WO 2010/019570 pamphlet, WO 2014/140180 pamphlet, WO 2015/116539 pamphlet, Examples include those described in Publication No. 2015/200119 pamphlet, International Publication No. 2016/028672 pamphlet, US Patent No. 9,244,059, and US Patent No. 9,505,839.

In one embodiment, the anti-LAG-3 antibody is an antibody that competes for binding to and/or binds to the same epitope on LAG-3 as one of the anti-LAG-3 antibodies described herein. be.

In one embodiment, the anti-LAG-3 inhibitor is a soluble LAG-3 protein, eg, IMP321 (Prima BioMed), eg, as disclosed in WO 2009/044273.

TIM-3 Inhibitors In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM-3. In some embodiments, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with a TIM-3 inhibitor for the treatment of a disease, such as cancer. It will be done. In some embodiments, the TIM-3 inhibitor is MGB453 (Novartis), LY3321367 (Eli Lilly), Sym023 (Symphogen), BGB-A425 (Beigene), INCAGN-2390 (Agenus/Incyte), MBS-9862 58( BMS/5 Prime), RO-7121661 (Roche), LY-3415244 (Eli Lilly), or TSR-022 (Tesaro).

Exemplary TIM-3 Inhibitors In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitors are those disclosed in U.S. Patent Application Publication No. 2015/0218274, published August 6, 2015, entitled "Antibody Molecules to TIM-3 and Uses Thereof." This is an anti-TIM-3 antibody molecule.

In one embodiment, the anti-TIM-3 antibody molecule is shown in Table 9 (e.g., from the ABTIM3-hum11 or ABTIM3-hum03 heavy chain and light chain variable region sequences disclosed in Table 9), or at least 1, 2, 3, 4, 5 or 6 complementarity determining regions (CDRs) (or all collectively CDR). In some embodiments, the CDRs follow the Kabat definition (eg, as described in Table 9). In some embodiments, the CDRs follow the Chothia definition (eg, as described in Table 9). In one embodiment, one or more of the CDRs (or all CDRs collectively) exhibit 1, 2, 3, 4, 5, 6 or more changes, e.g., in Table 9, or have amino acid substitutions (eg, conservative amino acid substitutions) or deletions to the amino acid sequence encoded by the nucleotide sequence shown in .

In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain variable region comprising a VHCDR1 amino acid sequence of SEQ ID NO: 189, a VHCDR2 amino acid sequence of SEQ ID NO: 190, and a VHCDR3 amino acid sequence of SEQ ID NO: 191, each disclosed in Table 9. (VH); and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 198, the VLCDR2 amino acid sequence of SEQ ID NO: 199, and the VLCDR3 amino acid sequence of SEQ ID NO: 200. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain variable region comprising a VHCDR1 amino acid sequence of SEQ ID NO: 189, a VHCDR2 amino acid sequence of SEQ ID NO: 208, and a VHCDR3 amino acid sequence of SEQ ID NO: 191, each disclosed in Table 9. (VH); and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO: 198, the VLCDR2 amino acid sequence of SEQ ID NO: 199, and the VLCDR3 amino acid sequence of SEQ ID NO: 200.

In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 194, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 194. In one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 204, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 204. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 210, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 210. In one embodiment, the anti-TIM-3 antibody molecule comprises the amino acid sequence of SEQ ID NO: 214, or a VL that includes an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 214. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 194 and a VL comprising the amino acid sequence of SEQ ID NO: 204. In one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 210 and a VL comprising the amino acid sequence of SEQ ID NO: 214.

In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 195, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 195. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 205, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 205. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 211, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 211. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO: 215, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 215. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 195 and a VL encoded by the nucleotide sequence of SEQ ID NO: 205. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO: 211 and a VL encoded by the nucleotide sequence of SEQ ID NO: 215.

In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 196, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 196. In one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 206, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 206. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 212, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 212. In one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 216, or an amino acid sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 216. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 196 and a light chain comprising the amino acid sequence of SEQ ID NO: 206. In one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 212 and a light chain comprising the amino acid sequence of SEQ ID NO: 216.

In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 197, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 197. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 207, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 207. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 213, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 213. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO: 217, or a nucleotide sequence at least 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 217. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 197 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 207. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 213 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 217.

The antibody molecules described herein can be produced by the vectors, host cells and methods described in US Patent Application Publication No. 2015/0218274.

Other Exemplary TIM-3 Inhibitors In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio/Tesaro). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of TSR-022, the heavy or light chain variable region sequences, or the heavy or light chain Contains arrays. In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences of APE5137 or APE5121 (or collectively all CDR sequences), heavy chain or light chain, e.g., as disclosed in Table 10. including variable region sequences, or heavy or light chain sequences. APE5137, APE5121 and other anti-TIM-3 antibodies are disclosed in WO 2016/161270.

In one embodiment, the anti-TIM-3 antibody molecule is antibody clone F38-2E2. In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of F38-2E2, the heavy or light chain variable region sequences, or the heavy or light chain Contains arrays.

In one embodiment, the anti-TIM-3 antibody molecule is LY3321367 (Eli Lilly). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of LY3321367, the heavy chain variable region sequence and/or the light chain variable region sequence, or the heavy chain sequence and/or light chain sequence.

In one embodiment, the anti-TIM-3 antibody molecule is Sym023 (Symphogen). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences of Sym023 (or all CDR sequences taken together), the heavy chain variable region sequence and/or the light chain variable region sequence, or the heavy chain sequence and/or light chain sequence.

In one embodiment, the anti-TIM-3 antibody molecule is BGB-A425 (Beigene). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of BGB-A425, the heavy chain variable region sequence and/or the light chain variable region sequence, or Contains heavy chain sequences and/or light chain sequences.

In one embodiment, the anti-TIM-3 antibody molecule is INCAGN-2390 (Agenus/Incyte). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or all CDR sequences collectively) of INCAGN-2390, the heavy chain variable region sequence and/or the light chain variable region sequence, or Contains heavy or light chain sequences.

In one embodiment, the anti-TIM-3 antibody molecule is BMS-986258 (BMS/5 Prime). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of BMS-986258, the heavy chain variable region sequence and/or the light chain variable region sequence, or Contains heavy chain sequences and/or light chain sequences.

In one embodiment, the anti-TIM-3 antibody or inhibitor molecule is RO-7121661 (Roche). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of the TIM-3 binding arm of RO-7121661, the heavy chain variable region sequence and/or the light chain chain variable region sequences, or heavy chain sequences and/or light chain sequences.

In one embodiment, the anti-TIM-3 antibody or inhibitor molecule is LY-3415244 (Eli Lilly). In one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the CDR sequences (or collectively all CDR sequences) of the TIM-3 binding arm of LY-3415244, the heavy chain variable region sequence and/or the light chain chain variable region sequences, or heavy chain sequences and/or light chain sequences.

Further known anti-TIM-3 antibodies include, for example, WO 2016/111947 pamphlet, WO 2016/071448 pamphlet, WO 2016/144803 pamphlet, and US Patent No. 8,552,156. US Pat. No. 8,841,418 and US Pat. No. 9,163,087.

In one embodiment, the anti-TIM-3 antibody is an antibody that competes for binding to and/or binds to the same epitope on TIM-3 as one of the anti-TIM-3 antibodies described herein. be.

Cytokines In yet another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure include, but are not limited to, interferon, IL-2, IL-15, IL- 7, or in combination with one or more cytokines including IL21. In certain embodiments, a TGFβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) is administered in combination with an IL-15/IL-15Ra complex. In some embodiments, the IL-15/IL-15Ra complex is selected from NIZ985 (Novartis), ATL-803 (Altor) or CYP0150 (Cytune).

Exemplary IL-15/IL-15Ra Complexes In one embodiment, the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15Ra). The IL-15/IL-15Ra complex can include IL-15 covalently or non-covalently bound to a soluble form of IL-15Ra. In certain embodiments, human IL-15 is non-covalently linked to a soluble form of IL-15Ra. In certain embodiments, the human IL-15 of the formulation has at least 85%, 90%, 95% the amino acid sequence of SEQ ID NO: 222 in Table 11, or SEQ ID NO: 222, as described in WO 2014/066527. %, or 99% or more, and the soluble form of human IL-15Ra is at least 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 223 in Table 11, or to SEQ ID NO: 223. Contains % or more identical amino acid sequences. The molecules described herein can be produced by the vectors, host cells and methods described in WO2007084342.

Other Exemplary IL-15/IL-15Ra Complexes In one embodiment, the IL-15/IL-15Ra complex comprises ALT-803, an IL-15/IL-15Ra Fc fusion protein (IL-15N72D: IL-15RaSu/Fc soluble complex). ALT-803 is described in WO 2008/143794. In one embodiment, the IL-15/IL-15Ra Fc fusion protein comprises a sequence as disclosed in Table 12.

In one embodiment, the IL-15/IL-15Ra complex comprises IL-15 fused to the sushi domain of IL-15Ra (CYP0150, Cytune). The sushi domain of IL-15Ra refers to a domain that begins with the first cysteine residue after the signal peptide of IL-15Ra and ends with the fourth cysteine residue after the signal peptide. Complexes of IL-15 fused to the sushi domain of IL-15Ra are described in WO 2007/04606 and WO 2012/175222. In one embodiment, the IL-15/IL-15Ra sushi domain fusion comprises a sequence as disclosed in Table 12.

In yet another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure contain Toll-like receptors (TLRs, e.g., TLR7, TLR8, TLR9) in combination with one or more agonists. In some embodiments, compounds of the present disclosure can be used in combination with a TLR7 agonist or TLR7 agonist conjugate.

In some embodiments, the TLR7 agonist comprises a compound disclosed in WO 2011/049677. In some embodiments, the TLR7 agonist is 3-(5-amino-2-(4-(2-(3,3-difluoro-3-phosphonopropoxy)ethoxy)-2-methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)propanoic acid. In some embodiments, the TLR7 agonist has the formula:

Contains compounds of

In another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure include one or more angiogenesis inhibitors, such as bevacizumab ( Avastin®), axitinib (Inlyta®); brivanib alaninate (BMS-582664, (S)-((R)-1-(4-(4-fluoro-2-methyl-1H-) indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); sorafenib (Nexavar® )); pazopanib (Votrient®); sunitinib malate (Sutent®); cediranib (AZD2171, CAS 288383-20-1); valgatef (BIBF1120, CAS 928326-83-4); foretinib ( GSK1363089); teratinib (BAY57-9352, CAS 332012-40-5; apatinib (YN968D1, CAS 811803-05-1); imatinib (Gleevec®); ponatinib (AP24534, CAS 943319-70-8) ); tivozanib (AV951, CAS 475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7); Batalanib dihydrochloride (PTK787, CAS 212141-51-0); Brivanib (BMS-540215, CAS 649735-46) -6); Vandetanib (Caprelsa® or AZD6474); Motesanib diphosphate (AMG706, CAS 857876-30-3, N-(2,3-dihydro-3,3-dimethyl-1H-indole-6) Dovitinib dilactic acid (TKI258, CAS 852433-84-2); Linfalib (ABT869, CAS 796967-16-3); cabozantinib (XL184, CAS 849217-68-1); lestaurtinib (CAS 111358-88-4), N-[5-[[[5-(1,1-dimethylethyl )-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS 345627-80-7); (3R,4R)-4-amino-1-((4-((3 -methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514), N-(3,4-dichloro-2-fluoro phenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolineamine (XL647, CAS 781613-23- 8); 4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]amino]-N-[3-(trifluoro methyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); or in combination with aflibercept (Eylea®).

When bevacizumab is used in combination with other therapeutic agents (such as TGFβ inhibitors and/or PD-1 inhibitors), it may be administered intravenously to the patient. For example, bevacizumab can be administered intravenously to a patient at a dose of 5 mg/kg. Bevacizumab may also be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, bevacizumab is administered at a dose of 5 mg/kg on days 1 and 15 of each cycle (eg, a 21-day or 28-day cycle).

In another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are one or more heat shock protein inhibitors, e.g. Spimycin (17-allylamino-17-demethoxygeldanamycin, also known as KOS-953 and 17-AAG, available from SIGMA and described in U.S. Pat. No. 4,261,989) ); letaspimycin (IPI504), ganetespib (STA-9090); [6-chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9H-purin-2-yl]amine ( BIIB021 or CNF2024, CAS 848695-25-0); trans-4-[[2-(aminocarbonyl)-5-[4,5,6,7-tetrahydro-6,6-dimethyl-4-oxo-3- (Trifluoromethyl)-1H-indazol-1-yl]phenyl]amino]cyclohexylglycine ester (SNX5422 or PF04929113, CAS 908115-27-5); 5-[2,4-dihydroxy-5-(1-methylethyl) ) phenyl]-N-ethyl-4-[4-(4-morpholinylmethyl)phenyl]-3-isoxazolecarboxamide (AUY922, CAS 747412-49-3); or 17-dimethylaminoethylamino-17 - used in combination with demethoxygeldanamycin (17-DMAG).

In yet another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more HDAC inhibitors or other epigenetic modulators. It will be done. Exemplary HDAC inhibitors include vorinostat (Zolinza®); romidepsin (Istodax®); trichostatin A (TSA); oxamflatin; vorinostat (Zolinza®), suberoylanilide hydroxamic acid); pyroxamide (siberoyl-3-aminopyridineamide hydroxamic acid); trapoxin A (RF-1023A); trapoxin B (RF-10238); cyclo[(αS,2S)-α-amino-η-oxo-2 -oxirane octanoyl-O-methyl-D-tyrosyl-L-isoleucyl-L-prolyl] (Cyl-1), cyclo[(αS,2S)-α-amino-η-oxo-2-oxirane octanoyl-O -Methyl-D-tyrosyl-L-isoleucyl-(2S)-2-piperidinecarbonyl] (Cyl-2), cyclic [L-alanyl-D-alanyl-(2S)-η-oxo-L-α-amino oxirane octanoyl-D-prolyl] (HC-toxin); cyclo[(αS,2S)-α-amino-η-oxo-2-oxirane octanoyl-D-phenylalanyl-L-leucyl-(2S)- 2-Piperidinecarbonyl] (WF-3161); Chlamydocin ((S)-cyclic (2-methylalanyl-L-phenylalanyl-D-prolyl-η-oxo-L-α-aminooxirane octanoyl); Apicidin ( Cyclo(8-oxo-L-2-aminodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-D-2-piperidinecarbonyl); romidepsin (Istodax®, FR-901228); 4-phenyl Butyrate; Spiricostatin A; Milproine (valproic acid); Entinostat (MS-275, N-(2-aminophenyl)-4-[N-(pyridin-3-yl-methoxycarbonyl)-amino-methyl ]-benzamide); depudecine (4,5:8,9-dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol; 4-(acetyl N1-(2-aminophenyl)-N8-phenyl-octanediamide (also known as BML-210); 4- (dimethylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide (also known as M344); (E)-3-(4-((2-(1H-indole-3- yl)ethyl)(2-hydroxyethyl)amino)-methyl)phenyl)-N-hydroxyacrylamide, panobinostat (Farydak®); mocetinostat and belinostat (also known as PXD101, Beleodaq®), or ( (2E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide), or chidamide (also known as CS055 or HBI-8000), (E)-N-(2-amino -5-fluorophenyl)-4-((3-(pyridin-3-yl)acrylamido)methyl)benzamide). Other epigenetic modulators include inhibitors of EZH2 (enhancer of zeste homolog 2), EED (embryonic ectoderm development), or LSD1 (lysine-specific histone demethylase 1A or KDM1A); Not limited to these.

In yet another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure include one or more indoleamine-pyrrole 2,3- Inhibitors of dioxygenases (IDO), such as indoximod (also known as NLG-8189), α-cyclohexyl-5H-imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919) ), or in combination with (4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine (also known as INCB024360) used.

Chimeric Antigen Receptors The present disclosure describes the use of chimeric antigen receptor (CAR) immune effector cells, such as T cells, or chimeric TCR-transduced immune effector cells, such as T cells, for use in combination with adoptive immunotherapy methods and reagents. An inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) is provided. This section briefly describes CAR technologies useful in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors), and describes CAR reagents, e.g., cells and compositions, and methods. Describe about.

In general, aspects of the present disclosure relate to or include isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), where the CAR is an antigen that binds to a tumor antigen as described herein. A binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment), a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular signaling domain (e.g., as described herein). intracellular signaling domain) (e.g., a costimulatory domain (e.g., a costimulatory domain as described herein) and/or a primary signaling domain (e.g., a primary signaling domain as described herein). In other aspects, the present disclosure includes a host cell comprising the nucleic acid described above and an isolated protein encoded by such a nucleic acid molecule. The proteins carried by the present invention, vectors containing them, host cells, pharmaceutical compositions, and administration and treatment methods are disclosed in detail in WO 2015142675.

In one aspect, the disclosure relates to an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR is a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein). A binding antigen binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment), a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular signaling domain (e.g., as described herein). intracellular signaling domains (e.g., costimulatory domains (e.g., costimulatory domains described herein) and/or primary signaling domains (e.g., primary signaling domains described herein) In some embodiments, the tumor-supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSCs). The disclosure features polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and/or polypeptides.

Instead, aspects of the present disclosure relate to isolated nucleic acids encoding chimeric T cell receptors (TCRs) comprising TCRα and/or TCRβ variable domains having specificity for the cancer antigens described herein. For example, Dembic et al. , Nature, 320, 232-238 (1986), Schumacher, Nat. Rev. Immunol. , 2, 512-519 (2002), Kershaw et al. , Nat. Rev. Immunol. , 5, 928-940 (2005), Xue et al. , Clin. Exp. Immunol. , 139, 167-172 (2005), Rossig et al. , Mol. Ther. , 10, 5-18 (2004) and Murphy et al. , Immunity, 22, 403-414 (2005); (Morgan et al. J. Immunol., 171, 3287-3295 (2003), Hughes et al., Hum. Gene Ther., 16, 1-16 (2005) , Zhao et al., J. Immunol., 174, 4415-4423 (2005), Roszkowski et al., Cancer Res., 65, 1570-1576 (2005) and Engels et al., Hum. r., 16 , 799-810 (2005); see US Patent Application Publication No. 2009/03046557. Such chimeric TCRs include, for example, MART-1, gp-100, p53 and NY-ESO-1, MAGE A3/ A6, MAGEA3, SSX2, HPV-16 E6 or HPV-16 E7. In other aspects, the present disclosure provides polypeptides encoded by such nucleic acids as well as such nucleic acids and/or polypeptides. A host cell comprising:

Non-limiting example sequences of various components that can be part of a CAR are listed in Table 11a, where "aa" represents an amino acid and "na" represents a nucleic acid encoding the corresponding peptide.

Targeting The present disclosure provides gRNA molecules or CRISPR systems as described herein that are further engineered to contain one or more CARs that direct immune effector cells to desired cells (e.g., cancer cells). cells containing or containing the same at any time, such as immune effector cells (eg, T cells, NK cells). This is accomplished through antigen-binding domains on CARs that are specific for cancer-associated antigens. Classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs of the present disclosure include: (1) cancer-associated antigens expressed on the surface of cancer cells; and (2) cancer-associated antigens that are themselves intracellular; There are two cancer-associated antigens, fragments (peptides) of such antigens are presented on the surface of cancer cells by the MHC (major histocompatibility complex).

In some embodiments, the tumor antigen is CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); or CLECL1); CD33; Epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1 ) Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor body 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor α (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testicin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PDGFR-β); stage-specific fetal antigen-4 (SSEA-4); CD20; folate receptor α; receptor tyrosine-protein kinase ERBB2 (Her2/neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrinB2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) Oncogene fusion protein consisting of (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp (1-1) Cer); Transglutaminase 5 (TGS5); High molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor β; Tumor endothelial marker 1 (TEM1/CD248); Tumor endothelial marker 7 related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Globo-H hexasaccharide moiety of glycoceramide (Globo-H); Breast differentiation antigen (NY-BR-1); Uroplakin 2 ( UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenoceptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 ( LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer/testis antigen 1 (NY-ESO-1); cancer/testis antigen 2 (LAGE-1a) ; melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin Binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53) p53 mutant; Prostein; Surviving; Telomerase; Prostate cancer tumor antigen-1 (PCTA-1 or Galectin-8), Melanoma antigen 1 recognized by T cells (Melan A or MART1); Rat sarcoma (Ras) Mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetyl gluco saminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog Family member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of imprinting sites) Squamous cell carcinoma antigen 3 (SART3) recognized by T cells; Paired box protein Pax-5 (PAX5); Proacrosin binding protein sp32 (OY-TES1); Lymphocyte-specific protein tyrosine kinase ( LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for late glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3) ; Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

The CARs described herein include an antigen-binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that binds a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein). be able to. In some embodiments, the tumor supporting antigen is an antigen present on stromal cells or myeloid-derived suppressor cells (MDSCs). Stromal cells can secrete growth factors to promote cell division within the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, CAR-expressing cells destroy tumor supporting cells, thereby indirectly inhibiting tumor growth or survival.

In embodiments, the stromal cell antigen is selected from one or more of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP), and tenascin. In certain embodiments, the FAP-specific antibody is, competes with, or has the same CDRs for binding as sibrotuzumab. In embodiments, the MDSC antigen is selected from one or more of CD33, CD11b, C14, CD15 and CD66b. Thus, in some embodiments, the tumor supporting antigen is one of bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15 and CD66b. Selected from the above.

Antigen Binding Domain Structure In some embodiments, the antigen binding domain of the encoded CAR molecule is an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single domain antibody (SDAB), VH or VL domain. , a camelid VHH domain, or a bifunctional (eg, bispecific) hybrid antibody (eg, Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).

In some instances, scFv can be prepared by methods known in the art (eg, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be created by linking the VH and VL regions together using a flexible polypeptide linker. The scFv molecule includes a linker of optimized length and/or amino acid composition (eg, a Ser-Gly linker). Linker length can greatly influence how the variable regions of a scFv fold and interact. In fact, the use of short polypeptide linkers (eg, 5-10 amino acids) prevents folding within the chain. Interchain folding is also required for the two variable regions to come together to form a functional epitope binding site. For examples of linker orientation and size, see, eg, Hollinger et al. 1993 Proc Natl Acad. Sci. U. S. A. 90:6444-6448, US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and WO 2006/020258 pamphlet and WO 2007/024715 Please refer to the issue pamphlet.

The scFv has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Linkers can include 19, 20, 25, 30, 35, 40, 45, 50 amino acid residues, or more. The linker sequence can include any naturally occurring amino acid. In some embodiments, the linker sequence includes the amino acids glycine and serine. In another embodiment, the linker sequence comprises a collection of glycine and serine repeats, such as ( _Gly4Ser )n, where n is a positive integer greater than or equal to 1 (SEQ ID NO: 232). In one embodiment, the linker can be (Gly ₄ Ser) ₄ (SEQ ID NO: 230) or (Gly ₄ Ser) ₃ (SEQ ID NO: 231). Differences in linker length may preserve or enhance activity, resulting in superior efficacy in activity tests.

In another aspect, the antigen binding domain is a T cell receptor (“TCR”), or a fragment thereof, such as a single chain TCR (scTCR). Methods for making such TCRs are known in the art. For example, WILLEMSEN Ra et al, GENE THERAPY 7: 1369-1377 (2000); ZHANG T -ET Al, Cancer Gene THER 11: 487-496 (2004); Aggen et al, GENE THER. 19(4):365-74 (2012). For example, an scTCR can be engineered that includes the Vα and Vβ genes from a T cell clone connected by a linker (eg, a flexible peptide). This approach is extremely useful for cancer-related targets, which are themselves intracellular, but where fragments (peptides) of such antigens are presented on the surface of cancer cells by MHC.

In certain embodiments, the encoded antigen binding domain has a binding affinity KD of 10 ⁻⁴ M to 10 ⁻⁸ M.

In one embodiment, the encoded CAR molecule comprises an antigen binding domain with a binding affinity KD for the target antigen of 10 ⁻⁴ M to 10 ⁻⁸ M, such as 10 ⁻⁵ M to 10 ⁻⁷ M, such as 10 ⁻⁶ M or 10 ⁻⁷ M. In one embodiment, the antigen binding domain has a binding affinity that is at least 5, 10, 20, 30, 50, 100 or 1,000 fold lower than a reference antibody, such as an antibody described herein. In one embodiment, the encoded antigen binding domain has a binding affinity that is at least 5 fold lower than a reference antibody (e.g., the antibody from which the antigen binding domain is derived). In one aspect, such antibody fragments are functional in that they effect a biological response, including, but not limited to, activation of an immune response, inhibition of signal transduction from its target antigen, inhibition of kinase activity, etc., as will be appreciated by one of skill in the art.

In one aspect, the antigen binding domain of the CAR is an scFv antibody fragment that is humanized compared to the murine sequence of the original scFv from which it is derived.

In one aspect, the antigen binding domain (eg, scFv) of a CAR of the present disclosure is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In one aspect, the entire CAR construct of the present disclosure is encoded by a nucleic acid molecule whose entire sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that there is a species-specific bias in the frequency of synonymous codons (ie, codons that code for the same amino acid) in coding DNA. Such codon degeneracy allows the same polypeptide to be encoded by different nucleotide sequences. Various codon optimization methods are known in the art, including those disclosed in US Pat. No. 5,786,464 and US Pat. No. 6,114,148.

Antigen binding domain (and targeted antigen)
In one embodiment, the antigen binding domain for CD19 is described, for example, in WO 2012/079000; WO 2014/153270; Kochenderfer, J. N. et al. , J. Immunother. 32(7), 689-702 (2009); Kochenderfer, J. N. , et al. , Blood, 116(20), 4099-4102 (2010); International Publication No. 2014/031687 pamphlet; Bejcek, Cancer Research, 55, 2346-2351, 1995; or US Patent No. 7,446,190 The described CAR is an antigen-binding portion of an antibody or antigen-binding fragment thereof, such as a CDR.

In one embodiment, the antigen-binding domain for mesothelin is an antigen-binding portion, eg a CDR, of an antibody, antigen-binding fragment or CAR, eg as described in WO 2015/090230. In one embodiment, the antigen-binding domain for mesothelin is, for example, WO 1997/025068, WO 1999/028471, WO 2005/014652, WO 2006/099141, WO 2009 /045957 pamphlet, 2009/068204 pamphlet, 2013/142034 pamphlet, 2013/040557 pamphlet, or 2013/063419 pamphlet of antibodies, antigen-binding fragments, or CARs described in Antigen binding portions, such as CDRs. In one embodiment, the antigen-binding domain for mesothelin is an antigen-binding portion, such as a CDR, of an antibody, antigen-binding fragment, or CAR described in WO 2015/090230.

In one embodiment, the antigen-binding domain for CD123 is an antigen-binding portion of an antibody, antigen-binding fragment or CAR, such as a CDR, as described, for example, in WO 2014/130635. In one embodiment, the antigen-binding domain for CD123 is, for example, WO 2014/138805, WO 2014/138819, WO 2013/173820, WO 2014/144622, WO 2001 /66139 pamphlet, 2010/126066 pamphlet, 2014/144622 pamphlet, or the antibody, antigen-binding fragment, or antigen-binding portion of CAR described in US Patent Application Publication No. 2009/0252742, For example, CDR. In one embodiment, the antigen-binding domain for CD123 is an antibody, antigen-binding fragment, or antigen-binding portion of a CAR, such as a CDR, as described in WO 2016/028896.

In one embodiment, the antigen-binding domain for EGFRvIII is an antigen-binding portion, eg, a CDR, of an antibody, antigen-binding fragment or CAR, eg, as described in WO 2014/130657.

In one embodiment, the antigen binding domain for CD22 is described, for example, in Haso et al. , Blood, 121(7):1165-1174 (2013); Wayne et al. , Clin Cancer Res 16(6):1894-1903 (2010); Kato et al. , Leuk Res 37(1):83-88 (2013); Creative BioMart (creativebiomart.net): MOM-18047-S(P).

In one embodiment, the antigen binding domain for CS-1 is the antigen binding portion of elotuzumab (BMS), such as the CDRs, as described, eg, by Tai et al. , 2008, Blood 112(4):1329-37; Tai et al. , 2007, Blood. 110(5):1656-63.

In one embodiment, the antigen binding domain for CLL-1 is an antibody available from R&D, ebiosciences, Abcam, such as PE-CLL1-hu Cat. No. 353604 (BioLegend); and PE-CLL1 (CLEC12A) Cat. No. 562566 ( BD), such as the CDRs. In one embodiment, the antigen-binding domain for CLL-1 is an antigen-binding portion, such as a CDR, of an antibody, antigen-binding fragment, or CAR described in WO 2016/014535.

In one embodiment, the antigen binding domain for CD33 is described, for example, in Bross et al. , Clin Cancer Res 7(6):1490-1496 (2001) (gemtuzumab ozogamicin, hP67.6), Caron et al. , Cancer Res 52(24):6761-6767 (1992) (lintuzumab, HuM195), Lapusan et al. , Invest New Drugs 30(3):1121-1131 (2012) (AVE9633), Aigner et al. , Leukemia 27(5):1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al. , Adv hematol 2012:683065 (2012) and Pizzitola et al. , Leukemia doi:10.1038/Lue. 2014.62 (2014), such as CDRs. In one embodiment, the antigen-binding domain for CD33 is an antibody, antigen-binding fragment, or antigen-binding portion of a CAR, such as a CDR, as described in WO 2016/014576.

In one embodiment, the antigen binding domain for GD2 is described, for example, in Mujoo et al. , Cancer Res. 47(4):1098-1104 (1987); Cheung et al. , Cancer Res 45(6):2642-2649 (1985), Cheung et al. , J Clin Oncol 5(9):1430-1440 (1987), Cheung et al. , J Clin Oncol 16(9):3053-3060 (1998), Handgrettinger et al. , Cancer Immunol Immunother 35(3):199-204 (1992). In some embodiments, the antigen binding domain for GD2 is selected from mAbs 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1 and 8H9. It is an antigen-binding portion of an antibody, for example, International Publication No. 2012033885 pamphlet, International Publication No. 2013040371 pamphlet, International Publication No. 2013192294 pamphlet, International Publication No. 2013061273 pamphlet, International Publication No. 2013123061 pamphlet, International Publication No. 2013074916 pamphlet and International Publication No. 201385552. Please refer to the issue pamphlet. In some embodiments, the antigen binding domain for GD2 is the antigen binding portion of an antibody described in US Patent Application Publication No. 20100150910 or WO2011160119.

In one embodiment, the antigen-binding domain for BCMA is, for example, the antigen-binding portion of an antibody described in WO2012163805, WO200112812 and WO2003062401, such as a CDR. In one embodiment, the antigen-binding domain for BCMA is an antigen-binding portion, such as a CDR, of an antibody, antigen-binding fragment, or CAR described in WO 2016/014565.

In one embodiment, the antigen binding domain for the Tn antigen is described, for example, in US Pat. No. 8,440,798, Brooks et al. , PNAS 107(22):10056-10061 (2010) and Stone et al. , OncoImmunology 1(6):863-873 (2012).

In one embodiment, the antigen binding domain for PSMA is described, for example, by Parker et al. , Protein Expr Purif 89(2): 136-145 (2013), US Patent Application Publication No. 20110268656 (J591 ScFv); Frigerio et al, European J Cancer 49(9): 2223-2232 (201 3) (scFvD2B ); antigen-binding portions, such as CDRs, of antibodies and single chain antibody fragments (scFv A5 and D7) described in WO 2006125481 pamphlet (mAb 3/A12, 3/E7 and 3/F11).

In one embodiment, the antigen binding domain for ROR1 is described, for example, in Hudecek et al. , Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847 pamphlet; and US Patent Application Publication No. 20130101607, such as antigen-binding portions, such as CDRs.

In one embodiment, the antigen binding domain for FLT3 is described, for example, in WO2011076922, US5777084, EP0754230, US20090297529. Antigen-binding portions of antibodies and some commercially available catalog antibodies (R&D, biosciences, Abcam), such as CDRs.

In one embodiment, the antigen binding domain for TAG72 is described, for example, in Hombach et al. , Gastroenterology 113(4):1163-1170 (1997); and antigen-binding portions, such as CDRs, of Abcam ab691.

In one embodiment, the antigen binding domain for FAP is described, for example, in Ostermann et al. , Clinical Cancer Research 14:4584-4592 (2008) (FAP5), US Patent Application Publication No. 2009/0304718; sibrotuzumab (e.g., Hofheinz et al., Oncology Research and Tre See atment 26(1), 2003. ); and Tran et al. , J Exp Med 210(6):1125-1135 (2013).

In one embodiment, the antigen binding domain for CD38 is daratumumab (see, e.g., Groen et al., Blood 116(21):1261-1262 (2010); MOR202 (e.g., U.S. Patent No. 8,263,746). or the antigen-binding portions of the antibodies described in US Pat. No. 8,362,211, such as the CDRs.

In one embodiment, the antigen binding domain for CD44v6 is described, for example, in Casucci et al. , Blood 122(20): 3461-3472 (2013).

In one embodiment, the antigen binding domain for CEA is described, for example, by Chmielewski et al. , Gastoenterology 143(4):1095-1107 (2012).

In one embodiment, the antigen binding domains for EPCAM include MT110, EpCAM-CD3 bispecific Ab (see, eg, clinicaltrials.gov/ct2/show/NCT00635596); edrecolumab; 3622W94; ING-1; and adecatumumab (MT201), such as CDRs.

In one embodiment, the antigen binding domain for PRSS21 is an antigen binding portion of an antibody described in US Pat. No. 8,080,650, such as a CDR.

In one embodiment, the antigen binding domain for B7H3 is the antigen binding portion, eg, CDR, of antibody MGA271 (Macrogenics).

In one embodiment, the antigen-binding domain for KIT is the antigen-binding portion of the antibodies described, e.g., in U.S. Pat. No. 7,915,391, U.S. Pat. It is CDR.

In one embodiment, the antigen binding domain for IL-13Ra2 is described, for example, in WO 2008/146911, WO 2004087758, some commercially available catalog antibodies and WO 2004087758. Antigen-binding portions of antibodies, such as CDRs.

In one embodiment, the antigen binding domain for CD30 is an antigen binding portion, eg a CDR, of an antibody as described, eg, in US Pat. No. 7,090,843 B1 and EP 0,805,871.

In one embodiment, the antigen binding domain for GD3 is described, for example, in US Pat. No. 7,253,263; US Pat. No. 8,207,308; WO 2005035577; and US Pat. No. 6,437,098.

In one embodiment, the antigen binding domain for CD171 is described, for example, in Hong et al. , J Immunother 37(2):93-104 (2014).

In one embodiment, the antigen binding domain for IL-11Ra is an antigen binding portion, eg, a CDR, of an antibody available from Abcam (Cat. No. ab55262) or Novus Biologicals (Cat. No. EPR5446). In another embodiment, the antigen binding domain for IL-11Ra is a peptide, eg, as described by Huang et al. , Cancer Res 72(1):271-281 (2012).

In one embodiment, the antigen binding domain for PSCA is described, for example, by Morgenroth et al. , Prostate 67(10):1121-1131 (2007) (scFv 7F5); Nejatollahi et al. , J of Oncology 2013 (2013), article ID 839831 (scFv C5-II);

In one embodiment, the antigen binding domain for VEGFR2 is described, for example, by Chinnasamy et al. , J Clin Invest 120(11):3953-3968 (2010).

In one embodiment, the antigen binding domain for LewisY is described, for example, by Kelly et al. , Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab (scFvs)); Dolezal et al. , Protein Engineering 16(1):47-56 (2003) (NC10 scFv).

In one embodiment, the antigen binding domain for CD24 is described, for example, in Maliar et al. , Gastroenterology 143(5):1375-1384 (2012).

In one embodiment, the antigen binding domain for PDGFR-β is an antigen binding portion, eg, a CDR, of antibody Abcam ab32570.

In one embodiment, the antigen binding domain for SSEA-4 is the antigen binding portion, eg, CDR, of antibody MC813 (Cell Signaling), or other commercially available antibodies.

In one embodiment, the antigen binding domain for CD20 is an antigen binding portion, eg, a CDR, of the antibodies rituximab, ofatumumab, ocrelizumab, veltuzumab, or GA101.

In one embodiment, the antigen binding domain for folate receptor alpha is antibody IMGN853, or as described in U.S. Patent Application Publication No. 20120009181; Antigen-binding portions of antibodies, such as CDRs.

In one embodiment, the antigen binding domain for ERBB2 (Her2/neu) is the antigen binding portion, eg, CDR, of the antibody trastuzumab, or pertuzumab.

In one embodiment, the antigen binding domain for MUC1 is an antigen binding portion, eg, a CDR, of antibody SAR566658.

In one embodiment, the antigen binding domain for EGFR is an antigen binding portion, eg, a CDR, of the antibody cetuximab, panitumumab, zaltumumab, nimotuzumab, or matuzumab.

In one embodiment, the antigen binding domain for NCAM is the antigen binding portion, eg, CDR, of antibody clone 2-2B: MAB5324 (EMD Millipore).

In one embodiment, the antigen binding domain for ephrinB2 is described, for example, in Abengozar et al. , Blood 119(19):4565-4576 (2012).

In one embodiment, the antigen binding domain for the IGF-I receptor is disclosed, for example, in US Pat. No. 8,344,112 B2; EP 2,322,550 A1; Antigen-binding portions of the antibodies described in US 2006/022995, such as CDRs.

In one embodiment, the antigen binding domain for CAIX is the antigen binding portion, eg, CDR, of antibody clone 303123 (R&D Systems).

In one embodiment, the antigen-binding domain for LMP2 is an antigen-binding portion, e.g., a CDR, of an antibody described, e.g., in U.S. Pat. .

In one embodiment, the antigen binding domain for gp100 is the antigen binding portion, e.g.

In one embodiment, the antigen binding domain for tyrosinase is an antigen binding portion, eg, a CDR, of an antibody described, eg, in US Pat. No. 5,843,674; or US Pat. No. 1,995,050,4048.

In one embodiment, the antigen binding domain for EphA2 is described, for example, in Yu et al. , Mol Ther 22(1):102-111 (2014).

In one embodiment, the antigen binding domain for GD3 is described, for example, in US Pat. No. 7,253,263; US Pat. No. 8,207,308; US Pat. A3 specification; US Patent Application Publication No. 20120276046; International Publication No. 2005035577; or the antigen-binding portion of an antibody described in US Patent No. 6,437,098, such as CDR.

In one embodiment, the antigen-binding domain for fucosyl GM1 is an antigen-binding portion, eg, a CDR, of an antibody described, eg, in US Patent Application Publication No. 20100297138; or WO 2007/067992.

In one embodiment, the antigen-binding domain for sLe is the antigen-binding portion, e.g. See Scott AM et al, Cancer Res 60:3254-61 (2000).

In one embodiment, the antigen binding domain for GM3 is an antigen binding portion, eg, a CDR, of antibody CA 2523449 (mAb 14F7).

In one embodiment, the antigen binding domain for HMWMAA is described, for example, in Kmiecik et al. , Oncoimmunology 3(1): e27185 (2014) (PMID: 24575382) (mAb9.2.27); US Pat. No. 6,528,481; International Publication No. 2010033866; Antigen-binding portions of the described antibodies, such as CDRs.

In one embodiment, the antigen binding domain for o-acetyl-GD2 is an antigen binding portion of antibody 8B6, eg, a CDR.

In one embodiment, the antigen binding domain for TEM1/CD248 is described, for example, in Marty et al. , Cancer Lett 235(2):298-308 (2006); Zhao et al. , J Immunol Methods 363(2):221-232 (2011).

In one embodiment, the antigen-binding domain for CLDN6 is an antigen-binding portion, eg, a CDR, of antibody IMAB027 (Ganymed Pharmaceuticals), eg, a clinical trial. See gov/show/NCT02054351.

In one embodiment, the antigen binding domain for TSHR is described, for example, in US Pat. No. 8,603,466; US Pat. No. 8,501,415; or US Pat. No. 8,309,693. Antigen-binding portions of antibodies described in, for example, CDRs.

In one embodiment, the antigen binding domain for GPRC5D is the antigen binding portion, eg, CDR, of antibody FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences).

In one embodiment, the antigen binding domain for CD97 is described, for example, in US Pat. No. 6,846,911; de Groot et al. , J Immunol 183(6):4127-4134 (2009); or antibodies from R&D: antigen binding portions of MAB3734, eg CDRs.

In one embodiment, the antigen binding domain for ALK is described, for example, by Mino-Kenudson et al. , Clin Cancer Res 16(5):1561-1571 (2010).

In one embodiment, the antigen binding domain for polysialic acid is described, for example, in Nagae et al. , J Biol Chem 288(47):33784-33796 (2013).

In one embodiment, the antigen binding domain for PLAC1 is described, for example, in Ghods et al. ,Biotechnol Appl Biochem 2013 doi:10.1002/bab. 1177, such as the CDRs.

In one embodiment, the antigen binding domain for Globo H is derived from antibody VK9; or for example from Kudryashov V et al, Glycoconj J. 15(3):243-9 (1998), Lou et al. , Proc Natl Acad Sci USA 111(7):2482-2487 (2014); MBr1: Bremer EG et al. J Biol Chem 259:14773-14777 (1984).

In one embodiment, the antigen binding domain for NY-BR-1 is described, for example, by Jager et al. , Appl Immunohistochem Mol Morphol 15(1):77-83 (2007).

In one embodiment, the antigen binding domain for WT-1 is described, eg, by Dao et al. , Sci Transl Med 5 (176): 176ra33 (2013); or the antigen-binding portion of the antibody described in WO 2012/135854 pamphlet, such as CDR.

In one embodiment, the antigen binding domain for MAGE-A1 is described, for example, by Willemsen et al. , J Immunol 174(12):7853-7858 (2005).

In one embodiment, the antigen binding domain for sperm protein 17 is described, for example, in Song et al. , Target Oncol 2013 Aug 14 (PMID: 23943313); Song et al. , Med Oncol 29(4):2923-2931 (2012).

In one embodiment, the antigen binding domain for Tie 2 is the antigen binding portion, eg, CDR, of antibody AB33 (Cell Signaling Technology).

In one embodiment, the antigen binding domain for MAD-CT-2 is an antigen binding portion, eg, a CDR, of an antibody described, eg, in PMID: 2450952; US Pat. No. 7,635,753.

In one embodiment, the antigen binding domain for Fos-related antigen 1 is an antigen binding portion, eg, a CDR, of antibody 12F9 (Novus Biologicals).

In one embodiment, the antigen binding domain for MelanA/MART1 is an antigen binding portion, eg a CDR, of an antibody described in EP 2514766 A2; or US Pat. No. 7,749,719.

In one embodiment, antigen binding domains for sarcoma translocation breakpoints are described, for example, in Luo et al, EMBO Mol. Med. 4(6):453-461 (2012), such as CDRs.

In one embodiment, the antigen binding domain for TRP-2 is described, for example, in Wang et al, J Exp Med. 184(6):2207-16 (1996), such as the CDRs.

In one embodiment, the antigen binding domain for CYP1B1 is an antigen binding portion, eg, a CDR, of an antibody described, eg, in Maecker et al, Blood 102(9):3287-3294 (2003).

In one embodiment, the antigen binding domain for RAGE-1 is the antigen binding portion, eg, CDR, of antibody MAB5328 (EMD Millipore).

In one embodiment, the antigen binding domain for human telomerase reverse transcriptase is the antigen binding portion, eg, CDR, of antibody catalog number: LS-B95-100 (Lifespan Biosciences).

In one embodiment, the antigen binding domain for intestinal carboxylesterase is the antigen binding portion, eg, CDR, of antibody 4F12: Catalog Number: LS-B6190-50 (Lifespan Biosciences).

In one embodiment, the antigen binding domain for mut hsp70-2 is the antigen binding portion, eg, CDR, of the antibody Lifespan Biosciences: Monoclonal: Catalog Number: LS-C133261-100 (Lifespan Biosciences).

In one embodiment, the antigen binding domain for CD79a is the anti-CD79a antibody [HM47/A9] (ab3121) available from Abcam; the antibody CD79A antibody #3351 available from Cell Signaling Technology; or the antibody available from Sigma Aldrich. antibody HPA017748 - the antigen-binding portion, eg, CDR, of an anti-CD79A antibody produced in rabbits.

In one embodiment, the antigen binding domain for CD79b is derived from the antibody polatuzumab vedotin, Dornan et al. , “Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymph homa”Blood. 2009 Sep 24;114(13):2721-9. doi:10.1182/blood-2009-02-205500. Anti-CD79b described in Epub 2009 Jul 24, or “4507 Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies"Abstracts of 56 ^th ASH Annual Meeting and Exposure, San The antigen-binding portion, such as the CDR, of the bispecific antibody anti-CD79b/CD3 described in Francisco, CA December 6-9 2014.

In one embodiment, the antigen binding domain for CD72 is as described in Myers, and Uckun, “An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 Jun; 18(1-2):119-22, or the antibody J3-109 described by Polson et al. , “Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection”Cancer Res March Anti-CD72 (10D6.8.1, mIgG1) antigen described in 15, 2009 69; 2358 A binding moiety, such as a CDR.

In one embodiment, the antigen binding domain for LAIR1 is the antigen binding portion, eg, CDR, of the antibody ANT-301 LAIR1 antibody available from ProSpec; or the anti-human CD305 (LAIR1) antibody available from BioLegend.

In one embodiment, the antigen binding domain for FCAR is manufactured by Sino Biological Inc. Antigen-binding portions, eg, CDRs, of the antibody CD89/FCAR antibody (Cat. No. 10414-H08H) available from Amazon.

In one embodiment, the antigen-binding domain for LILRA2 is the antigen-binding portion of the antibody LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or the mouse anti-LILRA2 antibody, monoclonal (2D7), available from Lifespan Biosciences, e.g. It is CDR.

In one embodiment, the antigen binding domain for CD300LF is the antibody mouse anti-CMRF35-like molecule 1 antibody, monoclonal [UP-D2] available from BioLegend, or the rat anti-CMRF35-like molecule 1 antibody, monoclonal [UP-D2] available from R&D Systems. 234903], such as CDRs.

In one embodiment, the antigen binding domain for CLEC12A is derived from the antibody Bispecific T cell Engager (BiTE) scFv antibody and Noordhuis et al. , “Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1xCD3 BiTE Antibody”53 ^rd ADC and MCLA-117 (Merus) described in ASH Annual Meeting and Exposure, December 10-13, 2011 Antigen binding portions, such as CDRs.

In one embodiment, the antigen binding domain for BST2 (also known as CD317) is the antibody mouse anti-CD317 antibody, monoclonal [3H4] available from Antibodies-Online or the mouse anti-CD317 antibody, monoclonal [696739] available from R&D Systems. Antigen binding portions, such as CDRs.

In one embodiment, the antigen binding domain for EMR2 (also known as CD312) is the antibody mouse anti-CD312 antibody, monoclonal [LS-B8033] available from Lifespan Biosciences, or the mouse anti-CD312 antibody, monoclonal [494025] available from R&D Systems. ], such as CDRs.

In one embodiment, the antigen binding domain for LY75 is the antibody mouse anti-lymphocyte antigen 75 antibody, monoclonal [HD30] available from EMD Millipore or the mouse anti-lymphocyte antigen 75 antibody, monoclonal [A15797] available from Life Technologies. antigen-binding portions, such as CDRs.

In one embodiment, the antigen binding domain for GPC3 is described by Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs. 2010 Nov; 21(10):907-916, or MDX-1414, HN3, or YP7, all three of which are described in Feng et al., “Glypican-3 antibodies: a new therapeutic target. for liver cancer.” FEBS Lett. 2014 Jan 21; 588 (2): 377-82), such as CDRs.

In one embodiment, the antigen binding domain for FCRL5 is described by Elkins et al. , “FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma”Mol Cancer Ther. 2012 Oct;11(10):2222-32, such as the CDR. In one embodiment, the antigen-binding domain for FCRL5 is, for example, WO 2001/038490, WO 2005/117986, WO 2006/039238, WO 2006/076691, The antigen-binding portion of the anti-FcRL5 antibody described in WO 2010/114940 pamphlet, WO 2010/120561 pamphlet, or WO 2014/210064 pamphlet, for example, CDR.

In one embodiment, the antigen binding domain for IGLL1 is the antibody mouse anti-immunoglobulin λ-like polypeptide 1 antibody available from Lifespan Biosciences, monoclonal [AT1G4], the mouse anti-immunoglobulin λ-like polypeptide 1 antibody available from BioLegend. , the antigen-binding portion of monoclonal [HSL11], such as the CDRs.

In one embodiment, the antigen binding domain comprises one, two, three (e.g., all three) heavy chain CDRs from the antibodies listed above, HC CDR1, HC CDR2 and HC CDR3 and/or the antibodies listed above. Includes one, two, three (eg, all three) light chain CDRs from the antibody, LC CDR1, LC CDR2, and LC CDR3. In one embodiment, the antigen binding domain comprises the heavy chain variable region and/or the variable light chain region of the antibodies listed above.

In another embodiment, the antigen binding domain comprises a humanized antibody or antibody fragment. In some embodiments, the non-human antibody is humanized, in which specific sequences or regions of the antibody are modified to increase their similarity to antibodies or fragments thereof that are naturally produced in humans. . In one aspect, the antigen binding domain is humanized.

In certain embodiments, a CAR, eg, an antigen binding domain of a CAR expressed by a cell of the present disclosure, binds CD19. CD19 is found on B cells throughout lineage differentiation, from the pro/pre-B cell stage to the terminally differentiated plasma cell stage. In certain embodiments, the antigen binding domain is a mouse scFv domain that binds human CD19, eg, the antigen binding domain of CTL019 (eg, SEQ ID NO: 252). In certain embodiments, the antigen binding domain is a humanized antibody or antibody fragment, eg, a scFv domain, derived from murine CTL019 scFv. In certain embodiments, the antigen binding domain is a human antibody or antibody fragment that binds human CD19. Exemplary scFv domains (and sequences thereof, eg, CDR, VL and VH sequences) that bind CD19 are provided in Table 12a. The scFv domain sequences provided in Table 12a include a light chain variable region (VL) and a heavy chain variable region (VH). VL and VH are joined by a linker comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 231), eg in the following orientation: VL-linker-VH.

The CDR sequences of the scFv domains of the CD19 antigen binding domain shown in Table 12a are shown in Table 12b for the heavy chain variable domain and in Table 12c for the light chain variable domain. "ID" represents the respective sequence number of each CDR.

In certain embodiments, the antigen binding domain comprises an anti-CD19 antibody, or a fragment thereof, such as a scFv. For example, the antigen binding domain includes the variable heavy chain and variable light chain listed in Table 12d. The linker sequence connecting the variable heavy chain and variable light chain can be any of the linker sequences described herein or can be GSTSGSGKPGSGEGSTKG (SEQ ID NO: 248). The light and heavy chain variable regions of the scFv can, for example, be in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In one embodiment, the CD19 binding domain comprises one or more (e.g., all three) light chain complementarity determining regions of the CD19 binding domains described herein, e.g., provided in Table 12a or Table 15. 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3) and/or as described herein, e.g. as provided in Table 12a or Table 16. One or more (e.g., all three) of the CD19 binding domain include heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3). )including. In one embodiment, the CD19 binding domain is one, two, or all of LC CDR1, LC CDR2, and LC CDR3 of any amino acid sequence as provided in Table 12c; and as provided in Table 12b. HC CDR1, HC CDR2 and HC CDR3 of any amino acid sequence as follows.

In the present disclosure, any known CD19 CAR in the art can be used in the construction of a CAR, such as the CD19 antigen binding domain of any known CD19 CAR. For example, LG-740; US Pat. No. 8,399,645; US Pat. No. 7,446,190; Xu et al. , Leuk Lymphoma. 2013 54(2):255-260 (2012); Cruz et al. , Blood 122(17):2965-2973 (2013); Brentjens et al. , Blood, 118(18): 4817-4828 (2011); Kochenderfer et al. , Blood 116(20):4099-102 (2010); Kochenderfer et al. , Blood 122(25): 4129-39 (2013); and the CD19 CAR described in 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10. In one embodiment, the antigen binding domain for CD19 is described, for example, in WO 2012/079000; WO 2014/153270; Kochenderfer, J. N. et al. , J. Immunother. 32(7), 689-702 (2009); Kochenderfer, J. N. , et al. , Blood, 116(20), 4099-4102 (2010); International Publication No. 2014/031687 pamphlet; Bejcek, Cancer Research, 55, 2346-2351, 1995; or US Patent No. 7,446,190 The described CAR is an antigen-binding portion of an antibody or antigen-binding fragment thereof, such as a CDR.

In certain embodiments, a CAR, eg, an antigen binding domain of a CAR expressed by a cell of the present disclosure, binds BCMA. BCMA is found to be preferentially expressed in mature B lymphocytes. In certain embodiments, the antigen binding domain is a mouse scFv domain that binds human BCMA. In certain embodiments, the antigen binding domain is a humanized antibody or antibody fragment, such as a scFv domain, that binds human BCMA. In certain embodiments, the antigen binding domain is a human antibody or antibody fragment that binds human BCMA. In embodiments, an exemplary BCMA CAR construct is created using the VH and VL sequences from WO 2012/0163805. In embodiments, further exemplary BCMA CAR constructs are created using the VH and VL sequences from WO 2016/014565. In embodiments, further exemplary BCMA CAR constructs are created using the VH and VL sequences from WO 2014/122144. In embodiments, further exemplary BCMA CAR constructs are created using the CAR molecules and/or VH and VL sequences from WO 2016/014789. In embodiments, additional exemplary BCMA CAR constructs are created using the CAR molecules and/or VH and VL sequences from WO 2014/089335. In embodiments, additional exemplary BCMA CAR constructs are created using the CAR molecules and/or VH and VL sequences from WO 2014/140248.

Any known BCMA CAR in the art can be used in this disclosure, such as the BMCA antigen binding domain of any known BCMA CAR. For example, those described herein.

Exemplary CAR Molecule In one aspect, a CAR, e.g., a CAR expressed by a cell of the present disclosure, is a CAR molecule comprising an antigen binding domain that binds a B cell antigen, e.g., as described herein, such as CD19 or BCMA. including.

In one embodiment, the CAR comprises a CD19 antigen-binding domain (e.g., a murine, human, or humanized antibody or antibody fragment that specifically binds CD19), a transmembrane domain, and an intracellular signaling domain (e.g., a costimulatory and/or intracellular signaling domains, including primary signaling domains).

Exemplary CAR molecules described herein are provided in Table 12e. The CAR molecules of Table 12e include the amino acid sequence of a CD19 antigen binding domain, such as any of the CD19 antigen binding domains provided in Table 12a.

In one aspect, a CAR, e.g., a CAR expressed by a cell of the present disclosure, comprises a CAR molecule that includes an antigen-binding domain that binds BCMA, e.g., a BCMA antigen-binding domain (e.g., specifically for BCMA, e.g., human BCMA). (a murine, human or humanized antibody or antibody fragment that binds), a transmembrane domain, and an intracellular signaling domain (eg, an intracellular signaling domain, including a costimulatory domain and/or a primary signaling domain).

Exemplary CAR molecules of the CARs described herein are provided in Table 1 of WO 2016/014565.

Transmembrane Domains Regarding transmembrane domains, in various embodiments a CAR can be designed to include a transmembrane domain attached to the extracellular domain of the CAR. A transmembrane domain includes one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 amino acids) and/or one or more additional amino acids related to the intracellular region of the protein from which the transmembrane protein is derived. (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is related to one of the other domains of the CAR, e.g., in one embodiment, the transmembrane domain is derived from a signaling domain, a costimulatory domain, or a hinge domain. It may be from the same protein that In another aspect, the transmembrane domain is not derived from the same protein from which any other domain of the CAR is derived. In some instances, transmembrane domains are configured such that, for example, interactions with other members of the receptor complex are avoided such that such domains are prevented from binding to transmembrane domains of the same or different surface membrane proteins. It can be selected or modified by amino acid substitutions to be minimal. In one aspect, the transmembrane domain has the ability to homodimerize with another CAR on the cell surface of a CAR-expressing cell. In different embodiments, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a natural binding partner present in the same CAR-expressing cell.

Transmembrane domains can be derived from either natural or recombinant sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain has the ability to signal one or more intracellular domains whenever the CAR binds to a target. Transmembrane domains particularly useful in this disclosure include, for example, the α, β or ζ chains of T cell receptors, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD4 9f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B 4), CD84 , CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME ( SLAMF8 ), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

In some examples, the transmembrane domain may be linked to the extracellular region of the CAR, eg, the antigen binding domain of the CAR, via a hinge, eg, a hinge from a human protein. For example, in one embodiment, the hinge is a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge. obtain. In one embodiment, the hinge or spacer comprises (eg, consists of) the amino acid sequence of SEQ ID NO: 265. In one aspect, the transmembrane domain comprises (eg, consists of) the transmembrane domain of SEQ ID NO: 266.

In certain embodiments, the encoded transmembrane domain is the amino acid sequence of the CD8 transmembrane domain, or the sequence having one, two, or more, but no more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO: 266. A sequence having at least 95% identity with the amino acid sequence number 266. In one embodiment, the encoded transmembrane domain comprises the sequence of SEQ ID NO: 266.

In other embodiments, the CAR-encoding nucleic acid molecule comprises a nucleotide sequence of a CD8 transmembrane domain comprising, for example, the sequence SEQ ID NO: 267 or SEQ ID NO: 304, or a sequence with at least 95% identity thereto.

In certain embodiments, the encoded antigen binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the encoded hinge region is at least 95% identical to the amino acid sequence of CD8 hinge, e.g. SEQ ID NO: 265; or the amino acid sequence of IgG4 hinge, e.g. SEQ ID NO: 268, or SEQ ID NO: 265 or SEQ ID NO: 268. Contains an array with . In other embodiments, the nucleic acid sequence encoding the hinge region is the sequence SEQ ID NO: 269 or SEQ ID NO: 270, corresponding to CD8 hinge or IgG4 hinge, respectively, or a sequence having at least 95% identity with SEQ ID NO: 269 or 270. including.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer has an amino acid sequence

including hinges. In some embodiments, the hinge or spacer is

contains a hinge encoded by the nucleotide sequence of

In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer is

Contains a hinge of amino acid sequence. In some embodiments, the hinge or spacer is

contains a hinge encoded by the nucleotide sequence of

In one aspect, the transmembrane domain may be recombinant, in which case the transmembrane domain will primarily contain hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine can be found at each end of the recombinant transmembrane domain.

Optionally, a short oligopeptide or polypeptide linker, 2-10 amino acids in length, can form a link between the transmembrane domain and the cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 273). In some embodiments, the linker is encoded by the nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 274).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Signaling Domains In embodiments of the present disclosure having intracellular signaling domains, such domains may contain one or more of, for example, a primary signaling domain and/or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain includes a primary signaling domain and a costimulatory signaling domain.

The intracellular signaling sequences within the cytoplasmic portion of the CARs of the present disclosure can be linked to each other randomly or in a fixed order. Optionally, short oligopeptide or polypeptide linkers, e.g., from 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length, are used between intracellular signaling sequences. can form a link. In one embodiment, a glycine-serine doublet may be used as a suitable linker. In one embodiment, a single amino acid, eg alanine, glycine, may be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed to include two or more, eg, 2, 3, 4, 5, or more costimulatory signaling domains. In certain embodiments, two or more, eg, 2, 3, 4, 5, or more costimulatory signaling domains are separated by a linker molecule, eg, a linker molecule described herein. In one embodiment, the intracellular signaling domain includes two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

Primary Signaling Domains Primary signaling domains regulate the primary activation of the TCR complex, either in a stimulating or inhibiting manner. A primary intracellular signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine activation motif or ITAM.

Examples of ITAM-containing primary intracellular signaling domains of particular use in this disclosure include CD3zeta, common FcRγ (FCER1G), FcγRIIa, FcRβ (Fc epsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10. and DAP12 domains. In one embodiment, a CAR of the present disclosure comprises an intracellular signaling domain, such as a CD3 zeta primary signaling domain.

In one embodiment, the encoded primary signaling domain comprises a functional signaling domain of CD3 zeta. The encoded CD3 zeta primary signaling domain has at least one, two or three modifications of the amino acid sequence of SEQ ID NO: 275 or SEQ ID NO: 276, but no more than 20, 10 or 5 modifications; Sequences having at least 95% identity to the amino acid sequence of SEQ ID NO: 275 or SEQ ID NO: 276 can be included. In some embodiments, the encoded primary signaling domain comprises the sequence of SEQ ID NO: 275 or SEQ ID NO: 276. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises the sequence of SEQ ID NO: 277, SEQ ID NO: 303, or SEQ ID NO: 278, or a sequence having at least 95% identity thereto.

Co-stimulatory Signaling Domains In some embodiments, the encoded intracellular signaling domain comprises a costimulatory signaling domain. For example, an intracellular signaling domain can include a primary signaling domain and a costimulatory signaling domain. In some embodiments, the encoded costimulatory signaling domains include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA- 1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1.ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CE ACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, Contains a functional signaling domain of a protein selected from one or more of LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, or NKG2D.

In certain embodiments, the encoded costimulatory signaling domain has at least one, two, or three modifications of the amino acid sequence of SEQ ID NO: 279 or SEQ ID NO: 280, but no more than 20, 10, or 5 modifications. or a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 279 or SEQ ID NO: 280. In one embodiment, the encoded costimulatory signaling domain comprises the sequence SEQ ID NO: 279 or SEQ ID NO: 280. In other embodiments, the nucleic acid sequence encoding the costimulatory signaling domain comprises the sequence of SEQ ID NO: 281, SEQ ID NO: 305, or SEQ ID NO: 282, or a sequence having at least 95% identity thereto.

In other embodiments, the encoded intracellular domain comprises the sequence of SEQ ID NO: 279 or SEQ ID NO: 280 and the sequence of SEQ ID NO: 275 or SEQ ID NO: 276, and the sequences comprising the intracellular signaling domain are, in the same frame, and is expressed as a single polypeptide chain.

In certain embodiments, the nucleic acid sequence encoding an intracellular signaling domain is a sequence of SEQ ID NO: 281, SEQ ID NO: 305, or SEQ ID NO: 282, or a sequence with at least 95% identity thereof, and a sequence of SEQ ID NO: 277, SEQ ID NO: 277. 306 or SEQ ID NO: 278, or a sequence having at least 95% identity thereto.

In some embodiments, the nucleic acid molecule further encodes a leader sequence. In one embodiment, the leader sequence comprises the sequence SEQ ID NO: 283.

In one aspect, the intracellular signaling domain is designed to include a CD3 zeta signaling domain and a CD28 signaling domain. In one aspect, the intracellular signaling domain is designed to include a CD3 zeta signaling domain and a 4-1BB signaling domain. In one aspect, the signal transduction domain of 4-1BB is the signal transduction domain of SEQ ID NO: 279. In one aspect, the CD3 zeta signaling domain is the signaling domain of SEQ ID NO: 275.

In one embodiment, the intracellular signaling domain is designed to include a CD3 zeta signaling domain and a CD27 signaling domain. In one aspect, the signal transduction domain of CD27 comprises the amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO: 280). In one aspect, the signaling domain of CD27 is

encoded by the nucleic acid sequence of

Vectors In another aspect, the present disclosure relates to vectors comprising nucleic acid sequences encoding CARs described herein. In one embodiment, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In one embodiment, the vector is a lentiviral vector. These vectors or portions thereof can be used, among other things, to generate template nucleic acids as described herein for use in CRISPR systems as described herein. Alternatively, vectors can be used to deliver nucleic acids directly to cells, eg, immune effector cells, eg, T cells, eg, allogeneic T cells, independently of the CRISPR system.

The present disclosure also provides vectors into which the DNA of the present disclosure is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. This also has the added advantage of low immunogenicity. A retroviral vector can also be a gamma retroviral vector, for example. A gamma retroviral vector contains, for example, a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g. two) long terminal repeats (LTRs) and a transgene of interest, e.g. a CAR. It may contain the encoding gene. Gamma retroviral vectors may lack viral structural genes such as gag, pol and env. Exemplary gamma retroviral vectors include murine leukemia virus (MLV), splenic focus forming virus (SFFV) and myeloproliferative sarcoma virus (MPSV) and vectors derived therefrom. For other gamma retroviral vectors, see, eg, Tobias Maetzig et al. , “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun;3(6):677-713.

In another embodiment, the vector comprising a nucleic acid encoding a desired CAR of the present disclosure is an adenoviral vector (A5/35). In another embodiment, expression of a nucleic acid encoding a CAR can be achieved using transposons such as sleeping beauty, crisper, CAS9 and zinc finger nucleases. June et al. below. 2009 Nature Reviews Immunology 9.10:704-716.

Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Disclosed herein are methods for making in vitro transcribed RNA CARs. The present disclosure also includes RNA constructs encoding CARs that can be directly transfected into cells. The method for generating mRNA used for transfection involves in vitro transcription (IVT) of the template with specially designed primers, followed by polyA addition to generate 3' and 5' untranslated sequences ("UTR"), 5' caps and It may involve the creation of a construct, typically 50-2000 bases long (SEQ ID NO: 310), containing an internal ribosome entry site (IRES), an expressed nucleic acid, and a poly A tail. RNA thus produced can efficiently transfect various types of cells. In one aspect, the template includes the sequence of a CAR.

Non-Viral Delivery Methods In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein to a cell or tissue or subject.

In some embodiments, non-viral methods include the use of transposons (also referred to as transposable elements). In some embodiments, a transposon is a piece of DNA that is capable of inserting itself into a location in the genome, e.g., a piece of DNA that has the ability to self-replicate and insert its copy into the genome, or more. A piece of DNA that can be spliced out from a long nucleic acid and inserted elsewhere in the genome. For example, a transposon includes a DNA sequence comprised of a transposed gene flanked by inverted repeat sequences.

In some embodiments, gene insertion using SBTS and nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas systems, or modified meganuclease reengineering homing ends) Cells, such as T or NK cells, that express the CARs described herein are created by using a combination of gene editing using nucleases).

In some embodiments, a cell of the present disclosure, e.g., a T or NK cell, e.g., an allogeneic T cell, e.g., as described herein (e.g., expressing a CAR described herein), A composition comprising (a) one or more gRNA molecules, e.g., as described herein, and one or more Cas molecules, e.g., Cas9 molecules, e.g., as described herein; , and (b) by contacting with a nucleic acid (such as a template nucleic acid molecule as described herein) comprising, for example, a CAR-encoding sequence as described herein. Without being bound by theory, it is believed that the composition of (a) above induces a breakpoint at or near the genomic DNA that is targeted by the targeting domain of one or more gRNA molecules. , and (b) is incorporated into the genome, eg, partially or completely, at or near the break point, and upon integration, the encoded CAR molecule will be expressed. In embodiments, expression of the CAR will be controlled by a promoter or other regulatory element endogenous to the genome (eg, a promoter that controls expression from a gene into which the nucleic acid of (b) has been inserted). In other embodiments, the nucleic acid of (b) comprises a promoter operably linked to the CAR-encoding sequence and/or other regulatory elements, eg, as described herein, such as the EF1-α promoter. Furthermore, upon integration, expression of the CAR will be controlled by its promoter and/or other regulatory elements. For further features of the present disclosure regarding the use of a CRISPR/Cas9 system, e.g., as described herein, to direct the incorporation of a nucleic acid sequence encoding a CAR, e.g., as described herein, see Other parts are described, for example in the sections on gene insertion and homologous recombination. In embodiments, the composition of a) above is a composition comprising an RNP comprising one or more gRNA molecules. In embodiments, RNPs containing gRNAs that target unique target sequences are introduced into cells simultaneously with, for example, a mixture of RNPs containing one or more gRNAs. In embodiments, RNPs containing gRNAs that target unique target sequences are introduced into cells sequentially.

In some embodiments, non-viral delivery methods allow reprogramming of cells, such as T or NK cells, and direct injection of cells into a subject. Advantages of non-viral vectors include, but are not limited to, ease and relatively low cost of production in sufficient quantities to address patient populations, stability during storage, and lack of immunogenicity. It will be done.

Promoter In one embodiment, the vector further comprises a promoter. In some embodiments, the promoter is selected from the EF-1 promoter, the CMV IE gene promoter, the EF-1α promoter, the ubiquitin C promoter, or the phosphoglycerate kinase (PGK) promoter. In one embodiment, the promoter is the EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO: 285.

Host Cells for CAR Expression As noted above, in some aspects the present disclosure provides host cells, e.g., immune effector cells, including a nucleic acid molecule, CAR polypeptide molecule, or vector as described herein, such as , a population of cells, e.g. a population of immune effector cells).

In certain aspects of the present disclosure, immune effector cells, such as T cells, can be obtained from a blood unit drawn from a subject using any technique known to those skilled in the art, such as Ficoll™ separation. In one preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells harvested by apheresis may be washed to remove the plasma fraction, and the cells may be placed in a suitable buffer or medium for optional subsequent processing steps. In one embodiment, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the cleaning solution may be calcium-free, magnesium-free, or may be free of many, if not all, divalent cations.

An initial activation step in the absence of calcium can lead to expanded activation. As will be readily understood by those skilled in the art, the washing step is performed using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 Cell Processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. This can be accomplished by methods known to those skilled in the art, such as by. After washing, cells can be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline with or without buffer. Alternatively, one can remove unwanted components of the apheresis sample and resuspend the cells directly in culture medium.

The method of the present application utilizes culture medium conditions containing up to 5% human AB serum, such as 2%, and uses known culture medium conditions and compositions, such as those described by Smith et al. , “Ex vivo expansion of human T cells for adaptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement “Clinical & Translational Immunology (2015) 4, e31; doi:10.1038/cti. It is recognized that those described in 2014.31 may be used.

In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, eg, by centrifugation on a PERCOLL™ gradient, or by countercurrent centrifugal elution.

The methods described herein are directed to specific subpopulations of immune effector cells, e.g., T cells, CD25+ depleted cells, e.g., regulatory T cell depleted populations, e.g., using negative selection techniques described herein. may include, for example, a selection of. Preferably, the population of regulatory T-depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

In one embodiment, regulatory T cells, eg, CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25 binding ligand, IL-2. In one embodiment, an anti-CD25 antibody, or a fragment thereof, or a CD25 binding ligand is conjugated to or otherwise coated on a substrate, such as a bead. In one embodiment, an anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein.

In one embodiment, regulatory T cells, eg, CD25+ T cells, are removed from the population using a CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 20 uL to 1e7 cells, or 15 uL to 1e7 cells, or 10 uL to 1e7 cells, or 5 uL to 1e7 cells, or 2.5 uL. 1e7 cells for 1.25 uL or 1e7 cells for 1.25 uL. In one embodiment, for example, more than 500 million cells/ml are used for regulatory T cell, eg, CD25+ depletion. In further embodiments, cell concentrations of 6, 7, 8, or 900 million cells/ml are used.

In one embodiment, the population of immune effector cells to be depleted comprises about 6 x ¹⁰⁹ CD25+ T cells. In other embodiments, the population of immune effector cells to be depleted comprises about 1×10 ⁹ to 1×10 ¹⁰ CD25+ T cells and any integer value therebetween. In one embodiment, the resulting population of regulatory T-depleted cells is 2 x 10 ⁹ regulatory T cells, e.g., CD25+ cells, or less (e.g., 1 x 10 ⁹ , 5 x 10 ⁸ , 1 x 10 ⁸ , 5×10 ⁷ , 1×10 ⁷ , or fewer CD25+ cells).

In one embodiment, regulatory T cells, eg, CD25+ cells, are removed from the population using a CliniMAC system with a depleted tubing set, eg, tubing 162-01. In one embodiment, the CliniMAC system is run in a depleted setting, such as DEPLATION 2.1.

While not wishing to be bound by any particular theory, reducing levels of immune cell negative regulators in a subject prior to apheresis or during manufacture of a CAR-expressing cell preparation (e.g., reducing unwanted immune cells, e.g., T _REG cells) ) may reduce a subject's risk of recurrence. For example, methods of depleting T _REG cells are known in the art. Methods of reducing T _REG cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

In some embodiments, the manufacturing method includes reducing the number of T _REG cells (eg, depleting them) prior to manufacturing the CAR-expressing cells. For example, the method of manufacturing comprises contacting a sample, e.g. an apheresis sample, with an anti-GITR antibody and/or an anti-CD25 antibody (or a fragment thereof, or a CD25-binding ligand), e.g. NK cells, NK cells) including depletion of T _REG cells prior to manufacturing the preparation.

In certain embodiments, the subject is pretreated with one or more therapies that reduce T _REG cells prior to cell harvest for CAR-expressing cell preparation, thereby reducing the subject's risk of relapse in response to CAR-expressing cell treatment. Ru. In certain embodiments, methods of reducing T _REG cells include, but are not limited to, administering to a subject one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or combinations thereof. . Administration of one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or combinations thereof can be performed before, during, or after injection of the CAR-expressing cell preparation.

In certain embodiments, the subject is pretreated with cyclophosphamide prior to cell harvest for CAR-expressing cell preparation, thereby reducing the subject's risk of relapse to CAR-expressing cell treatment. In certain embodiments, the subject is pretreated with an anti-GITR antibody prior to cell harvest for CAR-expressing cell preparation, thereby reducing the subject's risk of relapse to CAR-expressing cell treatment.

In one embodiment, the population of cells that are removed are not regulatory T cells or tumor cells, but cells that would otherwise adversely affect CART cell expansion and/or function, e.g., CD14, CD11b, CD33, CD15. , or other markers expressed by potentially immunosuppressive cells. In one embodiment, it is envisaged that such cells are removed simultaneously with regulatory T cells and/or tumor cells, or after said depletion, or in another order.

The methods described herein can include two or more selection steps, such as two or more depletion steps. Enrichment of T cell populations by negative selection can be achieved, for example, with a combination of antibodies directed against surface markers unique to the cells being negatively selected. One method is cell sorting and/or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8.

The methods described herein remove from a population cells expressing tumor antigens, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b that do not include tumor antigens, e.g., CD25, thereby removing CAR, For example, it may further include providing a population of regulatory T-depleted, eg, CD25+ depleted and tumor antigen-depleted cells suitable for expression of a CAR as described herein. In one embodiment, tumor antigen-expressing cells are removed simultaneously with regulatory T, eg, CD25+ cells. For example, the anti-CD25 antibody or fragment thereof and the anti-tumor antigen antibody or fragment thereof may be conjugated to the same substrate that can be used for cell removal, such as beads, or the anti-CD25 antibody or fragment thereof or the anti-tumor antigen Antibodies, or fragments thereof, can be attached to separate beads and mixtures thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells, eg, CD25+ cells, and the removal of tumor antigen-expressing cells are sequential, eg, can occur in either order.

Also, removing cells from a population that express checkpoint inhibitors, such as those described herein, such as one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells. Also provided are methods thereby comprising providing a population of regulatory T depletion, eg, CD25+ depleted cells and checkpoint inhibitor depleted cells, eg, PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, Includes CTLA-4, BTLA and LAIR1. In one embodiment, checkpoint suppressor expressing cells are removed at the same time as regulatory T, eg, CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be coupled to the same beads that can be used for cell removal, or an anti-CD25 antibody, or fragment thereof, and an anti-checkpoint inhibitor antibody, or fragment thereof, can be coupled to the same beads that can be used for cell removal. Suppressor antibodies, or fragments thereof, can be attached to separate beads and mixtures thereof can be used to remove cells. In other embodiments, the removal of regulatory T cells, eg, CD25+ cells, and the removal of checkpoint suppressor-expressing cells are sequential, eg, can occur in either order.

The methods described herein may include a positive selection step. For example, incubating with anti-CD3/anti-CD28 (e.g., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient for positive selection of the desired T cells. T cells can be isolated. In one embodiment, this time is about 30 minutes. In further embodiments, the time ranges from 30 minutes to 36 hours or more and all integer values in between. In further embodiments, this time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, this time is 10-24 hours, such as 24 hours. By using longer incubation times, only fewer T cells are present when compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. T cells can be isolated in any given situation. Furthermore, using longer incubation times may increase the capture efficiency of CD8+ T cells. Therefore, by simply leaving T cells bound to CD3/CD28 beads for a shorter or longer time and/or by increasing or decreasing the bead to T cell ratio (as further described herein). As described above), T cell subpopulations can be preferentially selected or counterselected at the beginning of culture or at other points in the process. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, T cell subpopulations can be preferentially selected or counterselected at the beginning of culture or other desired time points. can do.

In one embodiment, IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin or other suitable molecules, For example, one can select a T cell population that expresses one or more of the other cytokines. A screening method for cell expression can be determined, for example, by the method described in International Publication No. 2013/126712 pamphlet.

The concentrations of cells and surfaces (eg, particles such as beads) can be varied to isolate desired cell populations by positive or negative selection. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (e.g., increase the concentration of cells) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, or 5 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further aspect, a cell concentration of 75 million to 80 million, 85 million, 90 million, 95 million, or 100 million cells/ml is used. In further embodiments, a concentration of 125 million or 150 million cells/ml may be used.

By using high concentrations, cell yield, cell activation and cell expansion can be increased. Furthermore, using high cell concentrations may result in cells that may have weak expression of the target antigen of interest, such as CD28-negative T cells, or cells from samples where a large number of tumor cells are present (e.g., leukemia blood, tumor tissue, etc.). can be captured more efficiently. Such cell populations may be of therapeutic value and would be desirable to obtain. For example, using a high concentration of cells allows for more efficient selection of CD8+ T cells, which normally express weakly CD28.

In related embodiments, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This selects cells that express large amounts of the desired antigen to be bound to the particles. For example, CD4+ T cells highly express CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In one aspect, the concentration of cells used is 5 x ¹⁰⁶ /ml. In other embodiments, the concentration used can be about 1×10 ⁵ /ml to 1×10 ⁶ /ml and any integer value therebetween.

In other embodiments, cells can be incubated at various speeds for varying lengths of time either on a rotator at 2-10° C. or at room temperature.

T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step results in a more homogeneous formulation by removing granulocytes and some monocytes in the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and may be useful in this regard, one method includes PBS containing 20% DMSO and 8% human serum albumin, or 10% dextran. 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20 % human serum albumin and 7.5% DMSO or other suitable cell freezing medium containing, for example, Hespan and Plasmalyte A, and then the cells are frozen at a rate of 1° per minute. The sample is frozen at -80°C and stored in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods can be used as well as uncontrolled instant freezing at -20°C or in liquid nitrogen.

In certain embodiments, prior to activation using the methods of the present disclosure, cryopreserved cells are thawed and washed as described herein and left at room temperature for 1 hour.

Also contemplated in the context of this disclosure is the collection of blood samples or apheresis products from a subject at a time prior to the point at which expanded cells as described herein may be required. In this way, the source of cells to be expanded can be harvested at any time as needed and for any disease or disease state in which immune effector cell therapy, such as those described herein, may be beneficial. Desired cells, such as T cells, can be isolated and frozen for use in subsequent immune effector cell therapy. In one aspect, the blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing the disease, but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen and used at a later point in time. In certain embodiments, the sample is taken from the patient immediately after diagnosis of a particular disease as described herein, but prior to any treatment. In a further aspect, the cells are obtained from a blood sample or apheresis from a subject with drugs such as, but not limited to, natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, including treatment with antibodies or other immunoablative agents such as mycophenolate and FK506, such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolate, steroids, FR901228 and radiation. , isolated before any relevant treatment modality.

In a further aspect of the disclosure, the T cells are obtained directly from the patient after a treatment that leaves the subject with functional T cells. In this regard, after some cancer treatments, particularly with drugs that damage the immune system, the T cells obtained immediately after the treatment, usually during the period when the patient is recovering from the treatment, are It has been observed that the quality of the peptide is optimal or can be improved with respect to its expansion capacity ex vivo. Similarly, after ex vivo manipulation using the methods described herein, such cells may be in a state favorable for engraftment and enhanced expansion in vivo. Therefore, within the context of the present disclosure, it is contemplated that blood cells, including T cells, dendritic cells, or other hematopoietic cells, will be harvested during this recovery period. Additionally, in certain embodiments, mobilization (e.g., GM-CSF mobilization) and conditioning regimens are used to repopulate, recirculate, and regenerate specific cell types, particularly during defined time windows following treatment. and/or conditions favorable for expansion can be created in the subject. Exemplary cell types include T cells, B cells, dendritic cells and other immune system cells.

In one embodiment, immune effector cells expressing a CAR molecule, such as a CAR molecule described herein, are obtained from a subject who has received a low immunopotentiating dose of an mTOR inhibitor. In certain embodiments, the level of PD1-negative immune effector cells, e.g., T cells, or the ratio of PD1-negative immune effector cells, e.g., T cells/PD1-positive immune effector cells, e.g., T cells, in or recovered from the subject is at least Immune effector cells that are modified to express CAR, such as T A population of cells is collected.

In other embodiments, the population of immune effector cells, e.g., T cells, that has been or will be modified to express a CAR increases the number of PD1-negative immune effector cells, e.g., T cells, or or can be treated ex vivo by contacting with an amount of an mTOR inhibitor that increases the ratio of PD1 negative immune effector cells, eg, T cells/PD1 positive immune effector cells, eg, T cells.

In one embodiment, the T cell population is diaglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or protein, or cells in which DGK activity is reduced or inhibited. DGK-deficient cells can be created by genetic techniques, eg, by administering RNA interference agents, eg, siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with a DGK inhibitor described herein.

In one embodiment, the T cell population is Ikaros deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or cells in which Ikaros activity is reduced or inhibited, and Ikaros-deficient cells can be treated by genetic means, e.g., with RNA interference agents, e.g., siRNA. , shRNA, miRNA to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be created by treatment with an Ikaros inhibitor, such as lenalidomide.

In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, eg, does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficient cells may be generated by any of the methods described herein.

In certain embodiments, the NK cells are obtained from the subject. In another embodiment, the NK cell is a NK cell line, such as the NK-92 cell line (Conkwest).

In some aspects, the cells of the disclosure (e.g., immune effector cells of the disclosure, e.g., CAR-expressing cells of the disclosure) are induced pluripotent stem cells (“iPSCs”) or embryonic stem cells (ESCs); or T cells created from (eg, differentiated from) said iPSCs and/or ESCs. iPSCs can be generated from peripheral blood T lymphocytes, eg, peripheral blood T lymphocytes isolated from healthy volunteers, eg, by methods known in the art. Additionally, such cells can be differentiated into T cells by methods known in the art. For example, Themeli M. et al. , Nat. Biotechnol. , 31, pp. 928-933 (2013); doi:10.1038/nbt. 2678; see International Publication No. 2014/165707 pamphlet.

In another embodiment, the TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are listed in or cited in Table 13 for the treatment of cancer. Used in combination with one or more of the therapeutic agents listed in patents and patent applications. Each document listed in Table 13 includes all structural formulas within it.

Estrogen Receptor Antagonists In some embodiments, estrogen receptor (ER) antagonists in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) are used to treat diseases, such as cancer. is used. In some embodiments, the estrogen receptor antagonist is a selective estrogen receptor degrading agent (SERD). SERDs are estrogen receptor antagonists that bind to the receptor and cause, for example, degradation or downregulation of the receptor (Boer K. et al., (2017) Therapeutic Advances in Medical Oncology 9(7): 465-479 ). ER is, for example, a hormone-activated transcription factor important in the growth, development, and physiology of the human reproductive system. The ER is activated, for example, by the hormone estrogen (17β estradiol). ER expression and signaling has been implicated in cancer (eg, breast cancer), such as ER-positive (ER+) breast cancer. In some embodiments, the SERD is selected from LSZ102, fulvestrant, brillanestrant, or elacestrant.

Exemplary Estrogen Receptor Antagonists In some embodiments, the SERD comprises a compound disclosed in WO 2014/130310. In some embodiments, the SERD includes LSZ 102. LSZ102 has the chemical name: (E)-3-(4-((2-(2-(1,1-difluoroethyl)-4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3-yl) oxy)phenyl)acrylic acid.

Other Exemplary Estrogen Receptor Antagonists In some embodiments, the SERD comprises fulvestrant (CAS Registration Number: 129453-61-8), or a compound disclosed in WO 2001/051056. Fulvestrant is available from ICI 182780, ZM 182780, FASLODEX®, or (7α,17β)-7-{9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl}estra Also known as -1,3,5(10)-triene-3,17-diol. Fulvestrant is a high affinity estrogen receptor antagonist with an IC50 of 0.29 nM.

In some embodiments, the SERD comprises elacestrant (CAS Registration Number: 722533-56-4), or a compound disclosed in US Pat. No. 7,612,114. Elassestrant is RAD1901, ER-306323 or (6R)-6-{2-[ethyl({4-[2-(ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}-5, Also known as 6,7,8-tetrahydronaphthalen-2-ol. Elcestrant is an orally bioavailable, non-steroidal, complex selective estrogen receptor modulator (SERM) and SERD. Elassestrant is described, for example, by Garner F et al. , (2015) Anticancer Drugs 26(9):948-56.

In some embodiments, the SERD is Brylanestrant (CAS Registration Number: 1365888-06-7) or a compound disclosed in WO 2015/136017. Brilanestrant is GDC-0810, ARN810, RG-6046, RO-7056118 or (2E)-3-{4-[(1E)-2-(2-chloro-4-fluorophenyl)-1-( Also known as 1H-indazol-5-yl)but-1-en-1-yl]phenyl}prop-2-enoic acid. Brilanestrant is a next generation orally bioavailable selective SERD with an IC50 of 0.7 nM. Brilanestrants are, for example, those of Lai A. et al. (2015) Journal of Medicinal Chemistry 58(12):4888-4904.

In some embodiments, the SERD is described, for example, by McDonell et al. (2015) Journal of Medicinal Chemistry 58(12) 4883-4887. For other exemplary estrogen receptor antagonists, see, for example, WO 2011/156518, WO 2011/159769, WO 2012/037410, WO 2012/037411, and It is disclosed in Patent Application Publication No. 2012/0071535.

CDK4/6 Inhibitors In some embodiments, inhibitors of cyclin-dependent kinase 4 or 6 (CDK4/6) are used in combination with a TGFβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease, e.g., cancer. In some embodiments, the CDK4/6 inhibitor is selected from ribociclib, abemaciclib (Eli Lilly), or palbociclib.

Exemplary CDK4/6 Inhibitors In some embodiments, the CDK4/6 inhibitor is ribociclib (CAS Registration Number: 1211441-98-3), or US Pat. , 685,980.

In some embodiments, the CDK4/6 inhibitor is a compound disclosed in WO 2010/020675 and U.S. Patent Nos. 8,415,355 and 8,685,980. including.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS Registration Number: 1211441-98-3). Ribociclib is available as LEE011, KISQALI®, or 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H-pyrrolo[2, 3-d] Also known as pyrimidine-6-carboxamide.

Other Exemplary CDK4/6 Inhibitors In some embodiments, the CDK4/6 inhibitor comprises abemaciclib (CAS Registration Number: 1231929-97-7). Abemaciclib is LY835219 or N-[5-[(4-ethyl-1-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-1-(1-methylethyl )-1H-benzimidazol-6-yl]-2-pyrimidineamine. Abemaciclib is a CDK inhibitor selective for CDK4 and CDK6 and has been described, for example, by Torres-Guzman R et al. (2017) Oncotarget 10.18632/oncotarget. No. 17778.

In some embodiments, the CDK4/6 inhibitor comprises palbociclib (CAS Registration Number: 571190-30-2). Palbociclib is PD-0332991, IBRANCE® or 6-acetyl-8-cyclopentyl-5-methyl-2-{[5-(1-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d ] Also known as pyrimidin-7(8H)-one. Palbociclib inhibits CDK4 with an IC50 of 11 nM and CDK6 with an IC50 of 16 nM, as described, for example, by Finn et al. (2009) Breast Cancer Research 11(5):R77.

CXCR2 Inhibitors In some embodiments, chemokines (C-X-C motif ) Inhibitors of receptor 2 (CXCR2) are used. In some embodiments, the CXCR2 inhibitor is 6-chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2- selected from hydroxy-N-methoxy-N-methylbenzenesulfonamide, danilixin, reparixin, or navarixin.

Exemplary CXCR2 Inhibitors In some embodiments, the CXCR2 inhibitors are those described in U.S. Pat. Specification, US Patent Application Publication No. 2010/0152205, US 2011/0251205 and US 2011/0251206, and International Publication No. 2008/061740 pamphlet, US Patent Application Publication No. 2008/061741 pamphlet, 2008/062026 pamphlet, 2009/106539 pamphlet, 2010/063802 pamphlet, 2012/062713 pamphlet, 2013/168108 pamphlet, 2010/015613 pamphlet and Compounds disclosed in pamphlet No. 2013/030803. In some embodiments, the CXCR2 inhibitor is 6-chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2- Contains hydroxy-N-methoxy-N-methylbenzenesulfonamide or its choline salt. In some embodiments, the CXCR2 inhibitor is 6-chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2- Contains hydroxy-N-methoxy-N-methylbenzenesulfonamide choline salt. In some embodiments, the CXCR2 inhibitor is 2-hydroxy-N,N,N-trimethylethane-1-aminium 3-chloro-6-({3,4-dioxo-2-[(pentane-3- yl)amino]cyclobut-1-en-1-yl}amino)-2-(N-methoxy-N-methylsulfamoyl)phenolate (i.e., 6-chloro-3-((3,4-dioxo-2 -(pentan-3-ylamino)cyclobut-1-en-1-yl)amino)-2-hydroxy-N-methoxy-N-methylbenzenesulfonamide choline salt), with the following chemical structure:

has.

Other Exemplary CXCR2 Inhibitors In some embodiments, the CXCR2 inhibitor comprises Danilixin (CAS Registration Number: 954126-98-8). Danilixin is also known as GSK1325756 or 1-(4-chloro-2-hydroxy-3-piperidin-3-ylsulfonylphenyl)-3-(3-fluoro-2-methylphenyl)urea. For danilixin, see, for example, Miller et al. Eur J Drug Metab Pharmacokinet (2014) 39:173-181; and Miller et al. BMC Pharmacology and Toxicology (2015), 16:18.

In some embodiments, the CXCR2 inhibitor comprises reparixin (CAS Registration Number: 266359-83-5). Reparixin is also known as repertaxin or (2R)-2-[4-(2-methylpropyl)phenyl]-N-methylsulfonylpropanamide. Reparixin is a non-competitive allosteric inhibitor of CXCR1/2. Regarding reparixin, see, for example, Zarbock et al. Br J Pharmacol. 2008;155(3):357-64.

In some embodiments, the CXCR2 inhibitor comprises navarixin. Navarixin is MK-7123, SCH 527123, PS291822, or 2-hydroxy-N,N-dimethyl-3-[[2-[[(1R)-1-(5-methylfuran-2-yl)propyl]amino ]-3,4-Dioxocyclobuten-1-yl]amino]benzamide. For navarixin, see, for example, Ning et al. Mol Cancer Ther. 2012;11(6):1353-64.

CSF-1/1R Binders In some embodiments, CSF-1/1R binding agents are used in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) for the treatment of diseases, such as cancer. A 1R binder is used. In some embodiments, the CSF-1/1R binding agent is an inhibitor of macrophage colony stimulating factor (M-CSF), such as a monoclonal antibody or Fab to M-CSF (e.g., MCS110), a CSF-1R tyrosine kinase inhibitor. agents (e.g., 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide or BLZ945), receptor-type tyrosine selected from a kinase inhibitor (RTK) (eg, pexidartinib), or an antibody that targets CSF-1R (eg, emactuzumab or FPA008). In some embodiments, the CSF-1/1R inhibitor is BLZ945. In some embodiments, the CSF-1/1R binding agent is MCS110. In other embodiments, the CSF-1/1R binding agent is pexidartinib.

Exemplary CSF-1 Binding Agents In some embodiments, the CSF-1/1R binding agent comprises an inhibitor of macrophage colony stimulating factor (M-CSF). M-CSF is sometimes also known as CSF-1. In certain embodiments, the CSF-1/1R binding agent is an antibody to CSF-1 (eg, MCS110). In other embodiments, the CSF-1/1R binding agent is an inhibitor of CSF-1R (eg, BLZ945).

In some embodiments, the CSF-1/1R binding agent is a monoclonal antibody or Fab against M-CSF (e.g., MCS110/H-RX1), or WO 2004/045532 and WO 2005/068503. brochures (including H-RX1 or 5H4 (eg, antibody molecules or Fab fragments directed against M-CSF)) as well as binding agents to CSF-1 as disclosed in US Pat. No. 9,079,956.

In another embodiment, the CSF-1/1R binding agent is a CSF-1R tyrosine kinase inhibitor, 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazole- 6-yl)oxy)-N-methylpicolinamide (BLZ945), or WO 2007/121484 pamphlet and US Patent Nos. 7,553,854, 8,173,689 and US Pat. No. 8,710,048.

Other Exemplary CSF-1/1R Binders In some embodiments, the CSF-1/1R binder comprises pexidartinib (CAS Registry No. 1029044-16-3). Pexidartinib is PLX3397 or 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl ) Also known as pyridin-2-amine. Pexidartinib is a small molecule receptor tyrosine kinase (RTK) inhibitor of KIT, CSF1R and FLT3. FLT3, CSF1R and FLT3 are overexpressed or mutated in many cancer cell types and play a major role in tumor cell proliferation and metastasis. PLX3397 can bind to and inhibit the phosphorylation of stem cell factor receptor (KIT), colony stimulating factor-1 receptor (CSF1R) and Fms-like tyrosine kinase 3 (FLT3), which leads to the inhibition of tumor cell proliferation. It may result in inhibition and downregulation of macrophages, osteoclasts and mast cells involved in osteolytic metastatic disease.

In some embodiments, the CSF-1/1R binding agent is emactuzumab. Emactuzumab is also known as RG7155 or RO5509554. Emactuzumab is a humanized IgG1 mAb that targets CSF1R. In some embodiments, the CSF-1/1R binding agent is FPA008. FPA008 is a humanized mAb that inhibits CSF1R.

A2aR Antagonists In some embodiments, adenosine A2a receptor (A2aR) antagonists (in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) for the treatment of diseases, such as cancer For example, inhibitors of the A2aR pathway, eg, adenosine inhibitors, eg, inhibitors of A2aR or CD-73) are used. In some embodiments, the A2aR antagonist is PBF509 (NIR178) (Palobiofarma/Novartis), CPI444/V81444 (Corvus/Genentech), AZD4635/HTL-1071 (AstraZeneca/Hept ares), bipadenant (Redox/Juno), GBV- 2034 (Globavir), AB928 (Arcus Biosciences), theophylline, istradephylline (Kyowa Hakko Kogyo), Tozadenant/SYN-115 (Acorda), KW-6356 (Kyowa Hakko Kogyo), ST-4206 (Leadiant Biosciences) and Radenant/ Selected from SCH 420814 (Merck/Schering).

Exemplary A2aR Antagonists In some embodiments, the A2aR antagonist comprises PBF509 (NIR178) or a compound disclosed in US Pat. No. 8,796,284 or WO 2017/025918. PBF509 (NIR178) is also known as NIR178.

Other Exemplary A2aR Antagonists In certain embodiments, the A2aR antagonist comprises CPI444/V81444. CPI-444 and other A2aR antagonists are disclosed in WO 2009/156737. In certain embodiments, the A2aR antagonist is (S)-7-(5-methylfuran-2-yl)-3-((6-(((tetrahydrofuran-3-yl)oxy)methyl)pyridin-2- yl)methyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine. In certain embodiments, the A2aR antagonist is (R)-7-(5-methylfuran-2-yl)-3-((6-(((tetrahydrofuran-3-yl)oxy)methyl)pyridin-2- yl)methyl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine, or its racemic compound. In certain embodiments, the A2aR antagonist is 7-(5-methylfuran-2-yl)-3-((6-(((tetrahydrofuran-3-yl)oxy)methyl)pyridin-2-yl)methyl) -3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine.

In certain embodiments, the A2aR antagonist is AZD4635/HTL-1071. A2aR antagonists are disclosed in WO 2011/095625 pamphlet. In certain embodiments, the A2aR antagonist is 6-(2-chloro-6-methylpyridin-4-yl)-5-(4-fluorophenyl)-1,2,4-triazin-3-amine.

In certain embodiments, the A2aR antagonist is ST-4206 (Leadiant Biosciences). In certain embodiments, the A2aR antagonist is an A2aR antagonist described in US Pat. No. 9,133,197.

In certain embodiments, the A2aR antagonist is disclosed in U.S. Pat. The A2aR antagonist described in No.

In some embodiments, the A2aR antagonist is istradefylline (CAS Registry Number: 155270-99-8). Istradefylline is KW-6002 or 8-[(E)-2-(3,4-dimethoxyphenyl)vinyl]-1,3-diethyl-7-methyl-3,7-dihydro-1H-purine-2 , 6-dione. For istradefylline, see, for example, LeWitt et al. (2008) Annals of Neurology 63(3):295-302).

In some embodiments, the A2aR antagonist is Tozadenant (Biotie). Tozadenant is also known as SYN115 or 4-hydroxy-N-(4-methoxy-7-morpholin-4-yl-1,3-benzothiazol-2-yl)-4-methylpiperidine-1-carboxamide. Tozadenant blocks the effects of endogenous adenosine at A2a receptors, leading to enhanced dopamine effects at D2 receptors and inhibition of glutamate effects at mGluR5 receptors. In some embodiments, the A2aR antagonist is preladenant (CAS Registry Number: 377727-87-2). Preladenant is SCH 420814 or 2-(2-furanyl)-7-[2-[4-[4-(2-methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e] Also known as [1,2,4]triazolo[1,5-c]pyrimidin-5-amine. Preladenant was developed as a drug that acts as a potent and selective antagonist at the adenosine A2A receptor.

In some embodiments, the A2aR antagonist is bipadenant. Bipadenant is also known as BIIB014, V2006, or 3-[(4-amino-3-methylphenyl)methyl]-7-(furan-2-yl)triazolo[4,5-d]pyrimidin-5-amine . Other exemplary A2aR antagonists include, for example, ATL-444, MSX-3, SCH-58261, SCH-412,348, SCH-442,416, VER-6623, VER-6947, VER-7835, CGS-15943 and ZM-241,385.

In some embodiments, the A2aR antagonist is an A2aR pathway antagonist (eg, a CD-73 inhibitor, eg, an anti-CD73 antibody) and is MEDI9447. MEDI9447 is a monoclonal antibody specific for CD73. Targeting the extracellular production of adenosine by CD73 may reduce the immunosuppressive effects of adenosine. MEDI9447 was reported to have a variety of activities, including, for example, inhibition of CD73 ectonucleotidase activity, alleviation of AMP-mediated lymphocyte suppression and inhibition of syngeneic tumor growth. MEDI9447 can drive changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment. These changes include, for example, an increase in CD8 effector cells and activated macrophages and a decrease in the proportion of myeloid-derived suppressor cells (MDSCs) and regulatory T lymphocytes.

IDO Inhibitors In some embodiments, indoleamine 2,3-dioxygenase in combination with a TGFβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) is used to treat a disease, such as cancer. (IDO) and/or tryptophan 2,3-dioxygenase (TDO). In some embodiments, the IDO inhibitor is (4E)-4-[(3-chloro-4-fluoroanilino)-nitrosomethylidene]-1,2,5-oxadiazol-3-amine ( (also known as epacadostat or INCB24360), indoximod (), (1-methyl-D-tryptophan), α-cyclohexyl-5H-imidazo[5,1-a]isoindole-5-ethanol (also known as NLG919) , indoximod and BMS-986205 (formerly F001287).

Exemplary IDO Inhibitors In some embodiments, the IDO/TDO inhibitor is indoximod (New Link Genetics). Indoximod, the D isomer of 1-methyl-tryptophan, is an orally administered small molecule indoleamine 2,3-dioxygenase (IDO) pathway inhibitor that disrupts the mechanisms by which tumors escape immune-mediated destruction. be.

In some embodiments, the IDO/TDO inhibitor is NLG919 (New Link Genetics). NLG919 is a potent IDO (indoleamine-(2,3)-dioxygenase) pathway inhibitor with a Ki/EC50 of 7 nM/75 nM in cell-free assays.

In some embodiments, the IDO/TDO inhibitor is epacadostat (CAS Registration Number: 1204669-58-8). Epacadostat is also known as INCB24360 or INCB024360 (Incyte). Epacadostat is a potent and selective indoleamine 2,3-dioxygenase with an IC50 of 10 nM, which is highly selective compared to other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). (IDO1) inhibitor.

In some embodiments, the IDO/TDO inhibitor is F001287 (Flexus/BMS). F001287 is a small molecule inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1).

STING Agonists In some embodiments, STING agonists are used in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) to treat diseases, such as cancer. In some embodiments, the STING agonist is a cyclic dinucleotide, such as a cyclic dinucleotide comprising a purine or pyrimidine nucleobase (eg, adenosine, guanine, uracil, thymine, or cytosine nucleobase). In some embodiments, the nucleobases of the cyclic dinucleotide include the same nucleobase or different nucleobases.

In some embodiments, the STING agonist comprises an adenosine or guanosine nucleobase. In some embodiments, the STING agonist comprises one adenosine nucleobase and one guanosine nucleobase. In some embodiments, the STING agonist comprises two adenosine nucleobases or two guanosine nucleobases.

In some embodiments, the STING agonist comprises a modified cyclic dinucleotide that includes, for example, a modified nucleobase, a modified ribose, or a modified phosphate bond. In some embodiments, the modified cyclic dinucleotide includes a modified phosphate linkage, such as a thiophosphate.

In some embodiments, the STING agonist comprises a cyclic dinucleotide (eg, a modified cyclic dinucleotide) having a 2',5' or 3',5' phosphate linkage. In some embodiments, the STING agonist comprises a cyclic dinucleotide (eg, a modified cyclic dinucleotide) having an Rp or Sp stereochemistry for the phosphate bond.

In some embodiments, the STING agonist is MK-1454 (Merck). MK-1454 is a cyclic dinucleotide stimulator of interferon genes (STING) agonist that activates the STING pathway. Exemplary STING agonists are disclosed, for example, in WO 2017/027645.

Galectin Inhibitors In some embodiments, galectin inhibitors, e.g., galectin- 1 or galectin-3 inhibitors are used. In some embodiments, the combination includes a galectin-1 inhibitor and a galectin-3 inhibitor. In some embodiments, the combination includes a bispecific inhibitor (eg, a bispecific antibody molecule) that targets both galectin-1 and galectin-3. In some embodiments, the galectin inhibitor is an anti-galectin antibody molecule, GR-MD-02 (Galectin Therapeutics), Galectin-3C (Mandal Med), Anginex, or OTX-008 (OncoEthix, Merck) selected from. Galectins are a family of proteins that bind to β-galactosidase sugars.

The galectin protein family includes at least galectin-1, galectin-2, galectin-3, galectin-4, galectin-7, and galectin-8. Galectins, also called S-type lectins, are soluble proteins that have, for example, intracellular and extracellular functions.

Galectin-1 and galectin-3 are highly expressed in various tumor types. Galectin-1 and galectin-3 can promote angiogenesis and/or reprogram myeloid cells toward a tumor-promoting phenotype, eg, enhance immunosuppression from myeloid cells. Soluble galectin-3 can also bind to and/or inactivate infiltrating T cells.

Exemplary Galectin Inhibitors In some embodiments, the galectin inhibitor is an antibody molecule. In certain embodiments, the antibody molecule is a monospecific antibody molecule and binds a single epitope. For example, a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each binding the same epitope. In certain embodiments, the galectin inhibitor is an anti-galectin, eg, anti-galectin-1 or anti-galectin-3 antibody molecule. In some embodiments, the galectin inhibitor is an anti-galectin-1 antibody molecule. In some embodiments, the galectin inhibitor is an anti-galectin-3 antibody molecule.

In certain embodiments, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality is directed against a first epitope. and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In certain embodiments, the first and second epitopes overlap. In certain embodiments, the first and second epitopes do not overlap. In certain embodiments, the first and second epitopes are on different antigens, eg, different proteins (or different subunits of a multimeric protein). In certain embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In certain embodiments, the multispecific antibody molecule is a bispecific, trispecific, or tetraspecific antibody molecule.

In certain embodiments, the galectin inhibitor is a multispecific antibody molecule. In certain embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for two or fewer antigens. A bispecific antibody molecule comprises a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. It is characterized by In certain embodiments, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In certain embodiments, the first and second epitopes overlap. In certain embodiments, the first and second epitopes do not overlap. In certain embodiments, the first and second epitopes are on different antigens, eg, different proteins (or different subunits of a multimeric protein). In certain embodiments, a bispecific antibody molecule has a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope and that have binding specificity for a second epitope. A heavy chain variable domain sequence and a light chain variable domain sequence. In certain embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In certain embodiments, a bispecific antibody molecule comprises a half antibody, or a fragment thereof, that has binding specificity for a first epitope and a half antibody, or a fragment thereof, that has binding specificity for a second epitope. including fragments. In certain embodiments, the bispecific antibody molecule comprises an scFv, or fragment thereof, that has binding specificity for a first epitope and an scFv, or fragment thereof, that has binding specificity for a second epitope. including. In certain embodiments, the galectin inhibitor is a bispecific antibody molecule. In certain embodiments, the first epitope is located on galectin-1 and the second epitope is located on galectin-3.

Protocols for the production of bispecific or heterodimeric antibody molecules include, but are not limited to, the "knob in a hole" approach described in U.S. Pat. No. 5,731,168; Electrostatic steering Fc mating as described in WO 09/089004, WO 06/106905 and WO 2010/129304; for example, WO 07/110205 Strand Exchange Engineered Domain (SEED) heterodimer formation as described in, for example, WO 08/119353, WO 2011/131746 and WO 2013/ Fab arm exchange as described in brochure No. 060867; Double antibody conjugates by antibody cross-linking to generate heavy specificity structures; e.g., through reduction-oxidation cycles of the disulfide bond between the two heavy chains as described in U.S. Pat. No. 4,444,878. Also, bispecific antibody determinants produced by recombination of half antibodies (heavy chain-light chain pairs or Fabs) from different antibodies; functionalized antibodies, e.g. three Fab' fragments cross-linked through sulfhydryl reactive groups; e.g. biosynthetic binding proteins, e.g. as described in US Pat. No. 5,534,254, preferably through a disulfide tail; or pairs of scFv cross-linked through amine-reactive chemical cross-linking; e.g., bifunctional antibodies as described in U.S. Pat. No. 5,582,996; Fab fragments with different binding specificities dimerized through -fos and c-jun); bispecific and oligospecific monovalent and An oligovalent receptor, for example, two antibodies (two Fab VH-CH1 region of an antibody or Fab fragment via a bispecific DNA-antibody conjugate, e.g. a double-stranded piece of DNA, as described in US Pat. No. 5,635,602 Cross-linking; bispecific fusion proteins, e.g., expression constructs containing two scFvs with a hydrophilic helical peptide linker between them and a complete constant region, e.g., as described in U.S. Pat. No. 5,637,481; a first domain comprising a binding region of a multivalent and multispecific binding protein, e.g., an Ig heavy chain variable region, commonly referred to as a diabody, e.g., as described in U.S. Pat. No. 5,837,242; and a second domain comprising a binding region of an Ig light chain variable region (conformational structures creating bispecific, trispecific, or tetraspecific molecules) are also disclosed. further connected to the antibody hinge region and CH3 region with a peptide spacer that can dimerize to form bispecific/multivalent molecules, e.g., as described in U.S. Pat. No. 5,837,821; Minibody constructs with linked VL and VH chains; with short peptide linkers (e.g. 5 or 10 amino acids) in either orientation that can dimerize to form bispecific diabodies or VH and VL domains linked without any linker; trimers and tetramers, e.g., as described in U.S. Pat. No. 5,844,094; e.g., as described in U.S. Pat. No. 5,864,019 a string of VH domains (or VL domains in family members) connected by peptide bonds whose C-terminal bridging group is further associated with the VL domain to form a series of FVs (or scFv); and e.g. A single-chain binding polypeptide having both VH and VL domains connected by a peptide linker, as described in Patent No. 5,869,620, can be assembled into a multivalent structure through non-covalent or chemical cross-linking. combinations are known in the art, including, for example, those using both scFV-type or diabody-type formats to form homobivalent, heterobivalent, trivalent and tetravalent structures. Additional exemplary multispecific and bispecific molecules and methods of making them are described, for example, in U.S. Pat. No. 5,910,573, U.S. Pat. No. 5,932,448, U.S. Pat. Specification, U.S. Patent No. 6005079, U.S. Patent No. 6239259, U.S. Patent No. 6294353, U.S. Patent No. 6333396, U.S. Patent No. 6476198, U.S. Patent No. 6511663 , US Patent No. 6,670,453, US Patent No. 6,743,896, US Patent No. 6,809,185, US Patent No. 6,833,441, US Patent No. 7,129,330, US Patent No. 7,183,076, US Patent No. 7521056, U.S. Patent No. 7527787, U.S. Patent No. 7534866, U.S. Patent No. 7612181, U.S. Patent Application Publication No. 2002/004587A1, U.S. Patent Application Publication No. 2002/ 076406A1, US 2002/103345A1, US 2003/207346A1, US 2003/211078A1, US 2004/219643A1 Specification, US Patent Application Publication No. 2004/220388A1, US Patent Application Publication No. 2004/242847A1, US Patent Application Publication No. 2005/003403A1, US Patent Application Publication No. 2005/004352A1 , US Patent Application Publication No. 2005/069552A1, US Patent Application Publication No. 2005/079170A1, US Patent Application Publication No. 2005/100543A1, US Patent Application Publication No. 2005/136049A1, United States Patent Application Publication No. 2005/136051A1, US Patent Application Publication No. 2005/163782A1, US Patent Application Publication No. 2005/266425A1, US Patent Application Publication No. 2006/083747A1, US Patent Application Publication No. 2006/120960A1, U.S. Patent Application Publication No. 2006/204493A1, U.S. Patent Application Publication No. 2006/263367A1, U.S. Patent Application Publication No. 2007/004909A1, U.S. Patent Application Publication No. 2007/087381A1, US Patent Application Publication No. 2007/128150A1, US Patent Application Publication No. 2007/141049A1, US Patent Application Publication No. 2007/154901A1, US Patent Application Publication No. 2007/ 274985A1, US 2008/050370A1, US 2008/069820A1, US 2008/152645A1, US 2008/171855A1 Specification, US Patent Application Publication No. 2008/241884A1, US Patent Application Publication No. 2008/254512A1, US Patent Application Publication No. 2008/260738A1, US Patent Application Publication No. 2009/130106A1 , U.S. Patent Application Publication No. 2009/148905A1, U.S. Patent Application Publication No. 2009/155275A1, U.S. Patent Application Publication No. 2009/162359A1, U.S. Patent Application Publication No. 2009/162360A1, United States Patent Application Publication No. 2009/175851A1, US Patent Application Publication No. 2009/175867A1, US Patent Application Publication No. 2009/232811A1, US Patent Application Publication No. 2009/234105A1, US Patent Application Publication No. 2009/263392A1, US Patent Application Publication No. 2009/274649A1, European Patent Application No. 346087A2, International Publication No. 00/06605A2 pamphlet, International Publication No. 02/072635A2 pamphlet, International Publication No. 04/081051A1 pamphlet, International Publication No. 06/020258A2 pamphlet, International Publication No. 2007/044887A2 pamphlet, International Publication No. 2007/095338A2 pamphlet, International Publication No. 2007/137760A2 pamphlet, International Publication No. 2008/119353A1 International Publication No. 2009/021754A2 pamphlet, International Publication No. 2009/068630A1 pamphlet, International Publication No. 91/03493A1 pamphlet, International Publication No. 93/23537A1 pamphlet, International Publication No. 94/09131A1 pamphlet, International Publication No. This can be seen in pamphlets of International Publication No. 94/12625A2, pamphlets of International Publication No. 95/09917A1, pamphlets of International Publication No. 96/37621A2, and pamphlets of International Publication No. 99/64460A1.

In other embodiments, an anti-galectin antibody molecule, e.g., an anti-galectin-1 or anti-galectin-3 antibody molecule (e.g., a monospecific, bispecific, or multispecific antibody molecule), has another partner, For example, it is covalently linked, eg, fused, to a protein, eg, as a fusion molecule, eg, a fusion protein. In one embodiment, a bispecific antibody molecule has a first binding specificity for a first target (e.g., galectin-1) and a second binding specificity for a second target (e.g., galectin-3). It has sex.

The invention provides isolated nucleic acid molecules encoding the antibody molecules described above, vectors and host cells thereof. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

In some embodiments, the galectin inhibitor is a peptide, eg, a protein, that can bind to and inhibit the function of a galectin, eg, galectin-1 or galectin-3. In some embodiments, a galectin inhibitor is a peptide that can bind to and inhibit galectin-3 function. In some embodiments, the galectin inhibitor is the peptide galectin-3C. In some embodiments, the galectin inhibitor is a galectin-3 inhibitor disclosed in US Pat. No. 6,770,622.

Galectin-3C is an N-terminally truncated protein of galectin-3, and functions, for example, as a competitive inhibitor of galectin-3. Galectin-3C, for example, prevents endogenous galectin-3 from binding to laminin, which is on the surface of cancer cells, and other β-galactosidase glycoconjugates within the extracellular matrix (ECM). Galectin-3C and other exemplary galectin inhibiting peptides are disclosed in US Pat. No. 6,770,622.

In some embodiments, galectin-3C comprises the amino acid sequence of SEQ ID NO: 294, or substantially identical (eg, 90, 95 or 99%) identical amino acids thereto.

In some embodiments, a galectin inhibitor is a peptide that can bind to and inhibit galectin-1 function. In some embodiments, the galectin inhibitor is the peptide Anginex: Anginex is an anti-angiogenic peptide that binds to galectin-1 (Salomonsson E, et al., (2011) Journal of Biological Chemistry, 286(16):13801-13804). When Anginex binds to galectin-1, for example, the pro-angiogenic effect of galectin-1 can be interfered with.

In some embodiments, the galectin inhibitor, eg, galectin-1 or galectin-3 inhibitor, is a non-peptidic topomimetic molecule. In some embodiments, the non-peptide topomimetic galectin inhibitor is OTX-008 (OncoEthix). In some embodiments, the non-peptidic topomimetics are those disclosed in US Pat. No. 8,207,228. OTX-008, also known as PTX-008 or calixarene 0118, is a selective allosteric inhibitor of galectin-1. OTX-008 has the chemical name: N-[2-(dimethylamino)ethyl]-2-{[26,27,28-tris({[2-(dimethylamino)ethyl]carbamoyl}methoxy)pentacyclo[19. 3.1.1, 7.1, . 15,]octacosal-1(25),3(28),4,6,9(27),1012,15,17,19(26),21,23-dodecaen-25-yl]oxy}acetamide .

In some embodiments, the galectin, eg, galectin-1 or galectin-3 inhibitor, is a carbohydrate-based compound. In some embodiments, the galectin inhibitor is GR-MD-02 (Galectin Therapeutics).

In some embodiments, GR-MD-02 is a galectin-3 inhibitor. GR-MD-02 is, for example, a galactose-branched polysaccharide, also called galactoarabino-rhamnogalacturonate. GR-MD-02 and other galactose branched polymers, such as galactoarabino-rhamnogalacturonate, in U.S. Patent No. 8,236,780 and U.S. Patent Application Publication No. 2014/0086932 Disclosed.

MEK Inhibitors In some embodiments, MEK inhibitors are used in combination with TGFβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) to treat diseases, such as cancer. In some embodiments, the MEK inhibitor is selected from trametinib, selumetinib, AS703026, BIX 02189, BIX 02188, CI-1040, PD0325901, PD98059, U0126, XL-518, G-38963, or G02443714. In some embodiments, the MEK inhibitor is trametinib.

Exemplary MEK Inhibitors In some embodiments, the MEK inhibitor is trametinib. Trametinib is JTP-74057, TMT212, N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo- Also known as 3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenyl)acetamide, or Mekinist (CAS name 871700-17-3).

Other Exemplary MEK Inhibitors In some embodiments, the MEK inhibitor has the chemical name: (5-[(4-bromo-2-chlorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy) -1-Methyl-1H-benzimidazole-6-carboxamide.Selumetinib is also known as AZD6244 or ARRY 142886, for example as described in WO2003077914.

In some embodiments, the MEK inhibitor comprises AS703026, BIX 02189 or BIX 02188.

In some embodiments, the MEK inhibitor is 2-[(2-chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-, as described, for example, in WO 2000035436). Contains 3,4-difluoro-benzamide (also known as CI-1040 or PD184352).

In some embodiments, the MEK inhibitor is N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[, as described, for example, in WO 2002006213). Contains (2-fluoro-4-iodophenyl)amino]-benzamide (also known as PD0325901).

In some embodiments, the MEK inhibitor is manufactured by Biaffin GmbH & Co. , KG, Germany, including 2'-amino-3'-methoxyflavone (also known as PD98059).

In some embodiments, the MEK inhibitor is 2,3-bis[amino[(2-aminophenyl)thio]methylene]- Contains butane dinitrile (also known as U0126).

In some embodiments, the MEK inhibitor has CAS number 1029872-29-4 and is manufactured by ACC Corp. XL-518 (also known as GDC-0973) available from

In some embodiments, the MEK inhibitor comprises G-38963.

In some embodiments, the MEK inhibitor comprises G02443714 (also known as AS703206).

Further examples of MEK inhibitors are WO 2013/019906, WO 03/077914, WO 2005/121142, WO 2007/04415, WO 2008/024725. No. pamphlet and International Publication No. 2009/085983 pamphlet. Additional examples of MEK inhibitors include, but are not limited to, 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butane dinitrile (also known as U0126 and U.S. Pat. No. 2,779,780). (3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19 -tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione] (also known as E6201, described in WO 2003076424 pamphlet); Vemurafenib (PLX-4032, CAS 918504-65-1 ); (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7 (3H,8H)-dione (TAK-733, CAS 1035555-63-5); Pimasertib (AS-703026, CAS 1204531-26-9); 2-(2-fluoro-4-iodophenylamino)-N- (2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (AZD 8330); and 3,4-difluoro-2-[(2-fluoro-4-iodophenyl) )amino]-N-(2-hydroxyethoxy)-5-[(3-oxo-[1,2]oxazinan-2-yl)methyl]benzamide (CH4987655 specification or Ro4987655 specification).

c-MET Inhibitors In some embodiments, a c-MET inhibitor is used in combination with a TGFβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease, such as cancer. used. c-MET, a receptor tyrosine kinase that is overexpressed or mutated in many tumor cell types, plays a major role in tumor cell proliferation, survival, invasion, metastasis and tumor angiogenesis. Inhibiting c-MET can induce cell death in tumor cells that overexpress c-MET protein or express constitutively activated c-MET protein.

In some embodiments, the c-MET inhibitor is selected from capmatinib (INC280), JNJ-3887605, AMG 337, LY2801653, MSC2156119J, crizotinib, tivantinib, or golvatinib.

Exemplary c-MET Inhibitors In some embodiments, the c-MET inhibitor is capmatinib (INC280), or as described in U.S. Pat. Including the compounds described.

Other Exemplary c-MET Inhibitors In some embodiments, the c-MET inhibitor comprises JNJ-38877605. JNJ-38877605 is an orally administrable small molecule inhibitor of c-Met. JNJ-38877605 selectively binds to c-MET, thereby inhibiting c-MET phosphorylation and disrupting the c-Met signaling pathway.

In some embodiments, the c-Met inhibitor is AMG 208. AMG 208 is a selective small molecule inhibitor of c-MET. AMG 208 can inhibit ligand-dependent and ligand-independent activation of c-MET and inhibit its tyrosine kinase activity, thereby causing cell growth inhibition in tumors that overexpress c-Met.

In some embodiments, the c-Met inhibitor comprises AMG 337. AMG 337 is an orally bioavailable inhibitor of c-Met. AMG 337 selectively binds to c-MET, thereby disrupting the c-MET signaling pathway.

In some embodiments, the c-Met inhibitor comprises LY2801653. LY2801653 is an orally available small molecule inhibitor of c-Met. LY2801653 selectively binds to c-MET, thereby inhibiting c-MET phosphorylation and disrupting the c-Met signaling pathway.

In some embodiments, the c-Met inhibitor comprises MSC2156119J. MSC2156119J is an orally bioavailable inhibitor of c-Met. MSC2156119J selectively binds to c-MET, thereby inhibiting c-MET phosphorylation and disrupting c-Met-mediated signaling pathways.

In some embodiments, the c-MET inhibitor is capmatinib. Capmatinib is also known as INCB028060. Capmatinib is an orally bioavailable inhibitor of c-MET. Capmatinib selectively binds to c-Met, thereby inhibiting c-Met phosphorylation and disrupting the c-Met signaling pathway.

In some embodiments, the c-MET inhibitor comprises crizotinib. Crizotinib is also known as PF-02341066. Crizotinib is an orally available aminopyridine-based inhibitor of the receptor tyrosine kinases anaplastic lymphoma kinase (ALK) and c-Met/hepatocyte growth factor receptor (HGFR). Crizotinib binds to and inhibits ALK kinase and ALK fusion proteins in an ATP competitive manner. In addition, crizotinib inhibits c-Met kinase and disrupts the c-Met signaling pathway. Collectively, this drug inhibits tumor cell growth.

In some embodiments, the c-MET inhibitor comprises golvatinib. Golvatinib is an orally bioavailable dual kinase inhibitor of c-MET and VEGFR-2 with potential antineoplastic activity. Golvatinib binds to and inhibits both c-MET and VEGFR-2 activity, which can inhibit tumor cell growth and survival of tumor cells that overexpress these receptor tyrosine kinases. .

In some embodiments, the c-MET inhibitor is tivantinib. Tivantinib is also known as ARQ 197. Tivantinib is an orally bioavailable small molecule inhibitor of c-MET. Tivantinib binds to c-MET protein and disrupts the c-Met signaling pathway, leading to either overexpression of c-MET protein or expression of constitutively activated c-Met protein. Cell death can be induced in tumor cells.

IL-1β Inhibitors The interleukin-1 (IL-1) cytokine family is a group of secreted pleiotropic cytokines that have a central role in inflammatory and immune responses. Increased IL-1 is observed in multiple clinical settings including cancer (Apte et al. (2006) Cancer Metastasis Rev. p. 387-408; Dinarello (2010) Eur. J. Immunol. p. 599- 606). The IL-1 family includes, inter alia, IL-1 beta (IL-1b) and IL-1 alpha (IL-1a). IL-1b is elevated in lung, breast, and colorectal cancers (Voronov et al. (2014) Front Physiol. p. 114) and is associated with poor prognosis (Apte et al. (2000) Adv. Exp. Med. Biol. p. 277-88). While not wishing to be bound by theory, in some embodiments, secreted IL-1b derived from the tumor microenvironment and obtained by malignant cells is responsible in part for recruiting suppressive neutrophils. This is thought to promote tumor cell proliferation, increase invasiveness, and attenuate anti-tumor immune responses (Apte et al. (2006) Cancer Metastasis Rev. p. 387-408; Miller et al. (2007) J. Immunol. p. 6933-42). Experimental data show that inhibiting IL-1b reduces tumor burden and metastasis (Voronov et al. (2003) Proc. Natl. Acad. Sci. U.S.A. p. 2645-50) .

In some embodiments, interleukin-1 beta (IL-1β) inhibition in combination with a TGFβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) is used to treat a disease, such as cancer. agent is used. In some embodiments, the IL-1β inhibitor is selected from canakinumab, gevokizumab, anakinra, or rilonacept. In some embodiments, the IL-1β inhibitor is canakinumab.

Exemplary IL-1β Inhibitors In some embodiments, the IL-1β inhibitor is canakinumab. Canakinumab is also known as ACZ885 or ILARIS®. Canakinumab is a human monoclonal IgG1/κ antibody that neutralizes the biological activity of human IL-1β.

Canakinumab is disclosed, for example, in WO 2002/16436, US Patent No. 7,446,175, and European Patent No. 1313769. The heavy chain variable region of canakinumab is

(disclosed as SEQ ID NO: 1 in US Pat. No. 7,446,175). The light chain variable region of canakinumab is
MLPSQLIGFLLWVPASRGEIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAAAYYCHQSSSLPFTFGPGTKV It has the amino acid sequence of DIK (SEQ ID NO: 298) (disclosed as SEQ ID NO: 2 in US Pat. No. 7,446,175).

Canakinumab is useful, for example, in the treatment of cryopyrin-associated periodic syndrome (CAPS) in adults and children, in the treatment of systemic juvenile idiopathic arthritis (SJIA), in the symptomatic treatment of acute gouty arthritis attacks in adults, and in other IL-1β driven It is used to treat inflammatory diseases. Without wishing to be bound by theory, in some embodiments, an IL-1β inhibitor, e.g., canakinumab, inhibits, e.g., recruitment of immunosuppressive neutrophils to the tumor microenvironment, tumor angiogenesis. It is believed that anti-tumor immune responses can be increased by, for example, blocking one or more functions of IL-1b, including stimulating and/or promoting metastasis (Dinarello (2010) Eur. J. Immunol. p.599-606).

In some embodiments, the combinations described herein include an IL-1β inhibitor, canakinumab, or a compound disclosed in WO 2002/16436 and an inhibitor of an immune checkpoint molecule, e.g. inhibitors of PD-1 (eg, anti-PD-1 antibody molecules). IL-1 is a secreted pleiotropic cytokine that has a central role in inflammatory and immune responses. Increased IL-1 is observed in multiple clinical settings including cancer (Apte et al. (2006) Cancer Metastasis Rev. p. 387-408; Dinarello (2010) Eur. J. Immunol. p. 599- 606). IL-1b is elevated in lung, breast, and colorectal cancers (Voronov et al. (2014) Front Physiol. p. 114) and is associated with poor prognosis (Apte et al. (2000) Adv. Exp. Med. Biol. p. 277-88). While not wishing to be bound by theory, in some embodiments, secreted IL-1b derived from the tumor microenvironment and obtained by malignant cells is responsible in part for recruiting suppressive neutrophils. This is thought to promote tumor cell proliferation, increase invasiveness, and attenuate anti-tumor immune responses (Apte et al. (2006) Cancer Metastasis Rev. p. 387-408; Miller et al. (2007) J. Immunol. p. 6933-42). Experimental data show that inhibiting IL-1b reduces tumor burden and metastasis (Voronov et al. (2003) Proc. Natl. Acad. Sci. U.S.A. p. 2645-50) . Canakinumab can bind to IL-1b and inhibit IL-1-mediated signaling. Thus, in certain embodiments, an IL-1β inhibitor, e.g., canakinumab, enhances or enhances the immune-mediated antitumor effects of an inhibitor of PD-1 (e.g., an anti-PD-1 antibody molecule). used for.

In some embodiments, an IL-1β inhibitor, canakinumab, or a compound disclosed in WO 2002/16436 and an inhibitor of an immune checkpoint molecule, such as an inhibitor of PD-1 (e.g., an anti-PD -1 antibody molecules) are each administered at a dose and/or on a time schedule that, in combination, achieves the desired anti-tumor activity.

MDM2 Inhibitors In some embodiments, mouse double minute chromosome 2 homolog ( MDM2) inhibitors are used. The human homologue of MDM2 is also known as HDM2. In some embodiments, the MDM2 inhibitors described herein are also known as HDM2 inhibitors. In some embodiments, the MDM2 inhibitor is selected from HDM201 or CGM097.

In certain embodiments, the MDM2 inhibitor is (S)-1-(4-chlorophenyl)-7-isopropoxy-6-methoxy-2-( 4-(Methyl(((1r,4S)-4-(4-methyl-3-oxopiperazin-1-yl)cyclohexyl)methyl)amino)phenyl)-1,2-dihydroisoquinolin-3(4H)-one (also known as CGM097) or the compounds disclosed in WO 2011/076786 pamphlet). In one embodiment, the therapeutic agents disclosed herein are used in combination with CGM097.

In certain embodiments, the MDM2 inhibitor comprises an inhibitor of p53 and/or p53/Mdm2 interaction. In certain embodiments, the MDM2 inhibitor is (S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydropyridine) for the treatment of a disorder, such as a disorder described herein. -3-yl)-6-(4-chlorophenyl)-2-(2,4-dimethoxypyrimidin-5-yl)-1-isopropyl-5,6-dihydropyrrolo[3,4-d]imidazole-4( 1H)-one (also known as HDM201), or a compound disclosed in WO 2013/111105 pamphlet. In one embodiment, the therapeutic agents disclosed herein are used in combination with HDM201. In some embodiments, HDM201 is administered orally.

In one embodiment, the combinations disclosed herein are suitable for the treatment of cancer in vivo. For example, the combination can be used to inhibit the growth of cancerous tumors. The combinations may be used for the treatment of the disorders herein, including standard care treatments (e.g., for cancer or infectious disorders), vaccines (e.g., therapeutic cancer vaccines), cell therapy, radiotherapy, surgery or any other It may also be used in combination with one or more of the following therapeutic agents or modalities: For example, in order to achieve antigen-specific enhancement of immunity, the present combination can be administered together with the target antigen.

Pharmaceutical Compositions, Formulations, and Kits In another aspect, the present disclosure provides compositions comprising a combination described herein, formulated with a pharmaceutically acceptable carrier, e.g. Provides a composition with a unique composition. As used herein, "pharmaceutically acceptable carrier" includes any physiologically compatible solvent, dispersion medium, isotonic agent, absorption delaying agent, and the like. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).

The compositions described herein can be in various forms. This includes, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. The inhibitors described (including antibody inhibitors) may be in the form of an injectable or infusible solution. The mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In certain embodiments, the antibody is administered by intravenous infusion or injection. In another embodiment, the antibody is administered by intramuscular or subcutaneous injection.

The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, without limitation, intravenous administration. intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and Intrasternal injections and infusions are included.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high antibody concentrations. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. It can be prepared by Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, whereby the powder of the active ingredient plus any additional desired ingredients is prepared by pre-sterile filtering. arise from their solutions. The proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the necessary particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate and gelatin.

The combinations or compositions described herein are formulated into a formulation (e.g., an administration formulation or dosage form) suitable for administration (e.g., intravenous administration) to a subject as described herein. be able to. The formulations described herein can be liquid, lyophilized, or reconstituted formulations.

In certain embodiments, the formulation is a liquid formulation. In some embodiments, the formulation (eg, a liquid formulation) comprises a TGFβ inhibitor (eg, an anti-TGFβ antibody molecule as described herein) and a buffer. In some embodiments, the formulation (eg, a liquid formulation) comprises a PD-1 inhibitor (eg, an anti-PD-1 antibody molecule described herein) and a buffer. In some embodiments, the formulation (eg, a liquid formulation) comprises a PD-L1 inhibitor (eg, an anti-PD-L1 antibody molecule described herein) and a buffer. In some embodiments, the formulation (eg, a liquid formulation) includes a PD-L2 inhibitor (eg, an anti-PD-L2 antibody) and a buffer.

In some embodiments, the formulation (e.g., liquid formulation) is anti-TGF-β or anti-PD1 (or anti-PD1) as disclosed herein present at a concentration of about 25 mg/mL to about 250 mg/mL. -L1/L2) including antibody molecules. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 50 mg/mL to about 200 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 60 mg/mL to about 180 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 70 mg/mL to about 150 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 80 mg/mL to about 120 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 90 mg/mL to about 110 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 50 mg/mL to about 150 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 50 mg/mL to about 100 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 150 mg/mL to about 200 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 100 mg/mL to about 200 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 50 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 60 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 70 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 80 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 90 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 100 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 110 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 120 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 130 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 140 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 150 mg/mL. In some embodiments, the formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/L2) antibody molecules at a concentration of about 80 mg/mL to about 120 mg/mL, such as about 100 mg/mL.

In some embodiments, the formulation (eg, a liquid formulation) includes a buffer that includes histidine (eg, a histidine buffer). In certain embodiments, the buffer (e.g., histidine buffer) is about 1 mM to about 100 mM, such as about 2 mM to about 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, about 15 to about 25 mM, about 5mM to about 40mM, about 5mM to about 30mM, about 5mM to about 20mM, about 5mM to about 10mM, about 40mM to about 50mM, about 30mM to about 50mM, about 20mM to about 50mM, about 10mM to about 50mM, or about 5mM Present at a concentration of from to about 50 mM, such as about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the buffer (eg, histidine buffer) is present at a concentration of about 15 mM to about 25 mM, such as about 20 mM. In other embodiments, the buffer (eg, histidine buffer) has a pH of about 4 to about 7, such as about 5 to about 6, such as about 5, about 5.5, or about 6. In some embodiments, the buffer (eg, histidine buffer) has a pH of about 5 to about 6, such as about 5.5. In certain embodiments, the buffer comprises a histidine buffer at a concentration of about 15 mM to about 25 mM (eg, 20 mM) and has a pH of about 5 to about 6 (eg, 5.5). In certain embodiments, the buffer comprises histidine and histidine HCl.

In some embodiments, the formulation (e.g., liquid formulation) comprises antibody molecules as disclosed herein present at a concentration of 80-120 mg/mL, such as 100 mg/mL; histidine buffer at 15 mM 25mM (eg, 20mM) and a buffer having a pH of 5 to 6 (eg, 5.5).

In some embodiments, the formulation (eg, liquid formulation) further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is about 50mM to about 500mM, such as about 100mM to about 400mM, about 150mM to about 300mM, about 180mM to about 250mM, about 200mM to about 240mM, about 210mM to about 230mM, about 100mM to about 300mM, about 100mM to about 250mM, about 100mM to about 200mM, about 100mM to about 150mM, about 300mM to about 400mM, about 200mM to about 400mM, or about 100mM to about 400mM, e.g., about 100mM , about 150 mM, about 180 mM, about 200 mM, about 220 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM. In some embodiments, the formulation includes the carbohydrate or sucrose present at a concentration of about 200 mM to about 250 mM, such as about 220 mM.

In some embodiments, the formulation (e.g., liquid formulation) comprises antibody molecules as disclosed herein present at a concentration of 80-120 mg/mL, such as 100 mg/mL; histidine buffer at 15 mM a buffer present at a concentration of ~25mM (eg, 20mM) and having a pH of 5-6 (eg, 5.5); and a carbohydrate or sucrose present at a concentration of 200mM to 250mM, such as 220mM.

In some embodiments, the formulation (eg, liquid formulation) further includes a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20) is about 0.005% to about 0.1% (w/w), such as about 0.01% to about 0.08%, about 0.05% to about 0.1% (w/w). 02% to about 0.06%, about 0.03% to about 0.05%, about 0.01% to about 0.06%, about 0.01% to about 0.05%, about 0.01% to about 0.03%, about 0.06% to about 0.08%, about 0.04% to about 0.08%, or about 0.02% to about 0.08% (w/w), e.g. , about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about Present at a concentration of 0.09%, or about 0.1% (w/w). In some embodiments, the formulation includes a surfactant or polysorbate 20 present at a concentration of about 0.03% to about 0.05%, such as about 0.04% (w/w).

In some embodiments, the formulation (e.g., liquid formulation) comprises antibody molecules as disclosed herein present at a concentration of about 80-120 mg/mL, such as 100 mg/mL; a buffer comprising at a concentration of 15mM to 25mM (e.g. 20mM) and having a pH of 5 to 6 (e.g. 5.5); a carbohydrate or sucrose present at a concentration of 200mM to 250mM, e.g. 220mM; surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05%, such as 0.04% (w/w).

In some embodiments, a formulation (e.g., a liquid formulation) comprises an antibody molecule as disclosed herein present at a concentration of 100 mg/mL; a histidine buffer (e.g., histidine/histidine-HCL); a buffer having a pH of 5.5; a carbohydrate or sucrose present at a concentration of 220 mM; a surfactant or polysorbate 20 present at a concentration of 0.04% (w/w). including.

In some embodiments, liquid formulations are prepared by diluting a formulation containing an antibody molecule described herein. For example, a drug substance formulation can be diluted with a solution containing one or more excipients (eg, excipient concentrates). In some embodiments, the solution includes one, two, or all of histidine, sucrose, or polysorbate 20. In certain embodiments, the solution contains the same one or more excipients as the drug substance formulation. Exemplary excipients include, but are not limited to, amino acids (eg, histidine), carbohydrates (eg, sucrose), or surfactants (eg, polysorbate 20). In certain embodiments, the liquid formulation is not a reconstituted lyophilized formulation. In other embodiments, the liquid formulation is a reconstituted lyophilized formulation. In some embodiments, the formulation is stored as a liquid. In other embodiments, the formulation is prepared as a liquid and then stored after drying, eg, by lyophilization or spray drying.

In certain embodiments, for each container (e.g., vial) about 0.5 mL to about 10 mL (e.g., about 0.5 mL to about 8 mL, about 1 mL to about 6 mL, or about 2 mL to about 5 mL, e.g., about 1 mL). , about 1.2 mL, about 1.5 mL, about 2 mL, about 3 mL, about 4 mL, about 4.5 mL, about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, or about 10 mL) of the liquid formulation. In other embodiments, the liquid formulation is present in containers (e.g., vials) at least 1 mL (e.g., at least 1.2 mL, at least 1.5 mL, at least 2 mL, at least 3 mL, at least It is filled so that a removable volume of liquid formulation of 4 mL, or at least 5 mL) can be aspirated. In certain embodiments, liquid formulations are drawn undiluted from containers (eg, vials) at the clinical point. In certain embodiments, liquid formulations are diluted from drug substance formulations and withdrawn from containers (eg, vials) at a clinical point. In certain embodiments, the formulation (eg, a liquid formulation) is injected into an infusion bag, eg, within 1 hour (eg, within 45 minutes, 30 minutes, or 15 minutes) before beginning the infusion into the patient.

The formulations described herein can be stored in containers. Containers used for any one of the formulations described herein can include, for example, a vial and, optionally, a stopper, a cap, or both. In certain embodiments, the vial is a glass vial, such as a 6R white glass vial. In other embodiments, the stopper is a rubber stopper, such as a gray rubber stopper. In other embodiments, the cap is a flip-off cap, such as an aluminum flip-off cap. In some embodiments, the container includes a 6R white glass vial, a gray rubber stopper, and an aluminum flip-off cap. In some embodiments, the container (eg, vial) is a disposable container. In certain embodiments, there is from about 250 mg to about 1500 mg of antibody molecules as described herein within the container. In some embodiments, the container contains about 300 mg to about 1250 mg of antibody. In some embodiments, the container contains about 350 mg to about 1200 mg of antibody. In some embodiments, the container contains about 400 mg to about 1100 mg of antibody. In some embodiments, the container contains about 450 mg to about 1000 mg of antibody. In some embodiments, the container contains about 500 mg to about 900 mg of antibody. In some embodiments, the container contains about 600 mg to about 800 mg of antibody. In some embodiments, the container contains about 300 mg of antibody. In some embodiments, the container contains about 400 mg of antibody. In some embodiments, the container contains about 500 mg of antibody. In some embodiments, the container contains about 600 mg of antibody. In some embodiments, the container contains about 700 mg of antibody. In some embodiments, the container contains about 800 mg of antibody. In some embodiments, the container contains about 900 mg of antibody. In some embodiments, the container contains about 1000 mg of antibody.

In some embodiments, the formulation is a lyophilized formulation. In certain embodiments, lyophilized formulations are lyophilized or dried from liquid formulations comprising antibody molecules described herein. For example, each container (eg, vial) may be filled with about 1 to about 10 mL, such as about 6 to about 8 mL, of the liquid formulation and lyophilized.

In some embodiments, the formulation is a reconstituted formulation. In certain embodiments, reconstituted formulations are reconstituted from lyophilized formulations comprising antibody molecules described herein. For example, a reconstituted formulation can be prepared by dissolving the lyophilized formulation in a diluent so that the protein is dispersed in the reconstituted formulation. In some embodiments, the lyophilized formulation is reconstituted with about 1 mL to about 15 mL, such as about 5 mL to about 9 mL or about 7 mL of water or buffer for injection. In certain embodiments, the lyophilized formulation is reconstituted with about 6 mL to about 8 mL of water for injection, eg, at a clinical point.

In some embodiments, the reconstituted formulation comprises an antibody molecule (e.g., an anti-TGF-β or anti-PD-1 antibody (or anti-PD-L1/2) molecule as disclosed herein) and a buffer. including.

In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 25 mg/mL to about 250 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 50 mg/mL to about 200 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 antibody (or anti-PD-L1/2) molecules at a concentration of about 60 mg/mL to about 180 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 70 mg/mL to about 150 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 80 mg/mL to about 120 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 antibody (or anti-PD-L1/2) molecules at a concentration of about 90 mg/mL to about 110 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 50 mg/mL to about 150 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 50 mg/mL to about 100 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 150 mg/mL to about 200 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 100 mg/mL to about 200 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 50 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 60 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 70 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 80 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 90 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 100 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 110 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 120 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 130 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 140 mg/mL. In some embodiments, the reconstituted formulation comprises anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 150 mg/mL. In some embodiments, the reconstituted formulation contains anti-TGF-β or anti-PD1 (or anti-PD-L1/2) antibody molecules at a concentration of about 80 mg/mL to about 120 mg/mL, such as about 100 mg/mL. include.

In some embodiments, the reconstituted formulation includes a histidine-containing buffer (eg, a histidine buffer). In certain embodiments, the buffer (e.g., histidine buffer) is about 1 mM to about 100 mM, such as about 2 mM to about 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, about 15 to about 25 mM, about 5mM to about 40mM, about 5mM to about 30mM, about 5mM to about 20mM, about 5mM to about 10mM, about 40mM to about 50mM, about 30mM to about 50mM, about 20mM to about 50mM, about 10mM to about 50mM, or about 5mM Present at a concentration of from to about 50 mM, such as about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the buffer (eg, histidine buffer) is present at a concentration of about 15 mM to about 25 mM, such as about 20 mM. In other embodiments, the buffer (eg, histidine buffer) has a pH of about 4 to about 7, such as about 5 to about 6, such as about 5, about 5.5, or about 6. In some embodiments, the buffer (eg, histidine buffer) has a pH of about 5 to about 6, such as about 5.5. In certain embodiments, the buffer comprises a histidine buffer at a concentration of about 15 mM to about 25 mM (eg, 20 mM) and has a pH of about 5 to about 6 (eg, 5.5). In certain embodiments, the buffer comprises histidine and histidine HCl.

In some embodiments, the reconstituted formulation comprises antibody molecules as disclosed herein present at a concentration of about 80 to about 120 mg/mL, such as 100 mg/mL; histidine buffer at about 15 mM to about 100 mg/mL; a buffer at a concentration of about 25mM (eg, 20mM) and having a pH of 5-6 (eg, 5.5).

In some embodiments, the reconstituted formulation further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is 50mM to about 500mM, such as about 100mM to about 400mM, about 150mM to about 300mM, about 180mM to about 250mM, about 200mM to about 240mM, about 210mM to about 230mM, about 100mM to about 300mM, about 100mM to about 250mM, about 100mM to about 200mM, about 100mM to about 150mM, about 300mM to about 400mM, about 200mM to about 400mM, or about 100mM to about 400mM, e.g., about 100mM, Present at a concentration of about 15OmM, about 18OmM, about 20OmM, about 22OmM, about 25OmM, about 30OmM, about 35OmM, or about 40OmM. In some embodiments, the formulation includes the carbohydrate or sucrose present at a concentration of about 200 mM to about 250 mM, such as about 220 mM.

In some embodiments, the reconstituted formulation comprises antibody molecules disclosed herein present at a concentration of about 80 to about 120 mg/mL, such as 100 mg/mL; histidine buffer at about 15 mM to about 25 mM. a buffer present at a concentration of about 200 mM to about 250 mM, such as 220 mM; and a buffer having a pH of about 5 to about 6 (eg, 5.5); include.

In some embodiments, the reconstituted formulation further comprises a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20) is about 0.005% to about 0.1% (w/w), such as about 0.01% to about 0.08%, about 0.05% to about 0.1% (w/w). 02% to about 0.06%, about 0.03% to about 0.05%, about 0.01% to about 0.06%, about 0.01% to about 0.05%, about 0.01% to about 0.03%, about 0.06% to about 0.08%, about 0.04% to about 0.08%, or about 0.02% to about 0.08% (w/w), e.g. , about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about Present at a concentration of 0.09%, or about 0.1% (w/w). In some embodiments, the formulation includes a surfactant or polysorbate 20 present at a concentration of about 0.03% to about 0.05%, such as about 0.04% (w/w).

In some embodiments, the reconstituted formulation comprises antibody molecules as disclosed herein present at a concentration of about 80 to about 120 mg/mL, such as 100 mg/mL; histidine buffer at about 15 mM to about 100 mg/mL; a buffer comprising at a concentration of about 25mM (e.g., 20mM) and having a pH of about 5 to about 6 (e.g., 5.5); a carbohydrate or sucrose present at a concentration of about 200mM to about 250mM, e.g., 220mM; and a surfactant or polysorbate 20 present at a concentration of about 0.03% to about 0.05%, for example 0.04% (w/w).

In some embodiments, the reconstituted formulation comprises antibody molecules as disclosed herein present at a concentration of 100 mg/mL; and a histidine buffer (e.g., histidine/histidine-HCL) at a concentration of 20 mM. a buffer having a pH of 5.5; a carbohydrate or sucrose present at a concentration of 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.04% (w/w).

In some embodiments, the formulation is delivered at least 1 mL (e.g., at least 1.2 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, Aspirate a drawable volume of reconstituted formulation of 4 mL, 4.5 mL, 5 mL, 5.5 mL, 6 mL, 6.5 mL, 7 mL, 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL or 10 mL). be reconfigured so that it can be In certain embodiments, the formulation is reconstituted and/or withdrawn from the container (eg, vial) at the clinical point. In certain embodiments, the formulation (eg, reconstituted formulation) is injected into an infusion bag, eg, within 1 hour (eg, within 45 minutes, 30 minutes, or 15 minutes) prior to initiation of infusion into the patient.

In some embodiments, the reconstituted formulation has a fill volume of about 1 mL to about 5 mL. In certain embodiments, the reconstituted formulation has a fill volume of about 2 to about 4 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.2 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.4 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.6 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.8 mL.

Other exemplary buffers that may be used in the formulations described herein include, but are not limited to, arginine buffer, citrate buffer, or phosphate buffer. Other exemplary carbohydrates that may be used in the formulations described herein include, but are not limited to, trehalose, mannitol, sorbitol, or combinations thereof. The formulations described herein may also contain tonicity agents, such as sodium chloride, and/or stabilizers, such as amino acids such as glycine, arginine, methionine, or combinations thereof.

Although antibody molecules may be administered by a variety of methods known in the art, for many therapeutic applications the preferred route/mode of administration is intravenous injection or infusion. For example, antibody molecules may be administered at a dose of about 35 to 440 mg/m ² , typically about 70 mg/m ² to about 310 mg/m ² , and more typically about 110 mg/m ² to about 130 mg/m ² may be administered by intravenous infusion at a rate of greater than 20 mg/min, such as from 20 to 40 mg/min, and typically 40 mg/min or more. In embodiments, the antibody molecules are about 1 mg/m ² to about 100 mg/m ² , preferably about 5 mg/m ² to about 50 mg/m ² , about 7 mg/m ² to about 25 mg/m ² and more preferably, It may be administered by intravenous infusion at a rate of less than 10 mg/min; preferably 5 mg/min or less to reach a dose of about 10 mg/ ^m2 . As one of ordinary skill in the art will appreciate, the route and/or mode of administration will vary depending on the desired result. In certain embodiments, the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for preparing such formulations are patented or generally known to those skilled in the art. For example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed. , Marcel Dekker, Inc. , New York, 1978.

In certain embodiments, antibody molecules can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into a tablet, or incorporated directly into the subject's diet. For oral therapeutic administration, the compound can be incorporated with excipients and used in the form of orally ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In order to administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material that prevents its inactivation. The therapeutic compositions can also be administered with medical devices known in the art.

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as the exigencies of the therapeutic situation dictate. good. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unit dosages to the subject under treatment; each unit being associated with the necessary pharmaceutical carriers and containing the desired contains a predetermined amount of active compound calculated to produce a therapeutic effect. The specifications for the dosage unit form of the present invention depend on (a) the unique characteristics of the active compound and the detailed therapeutic effect sought to be achieved, and (b) the technology of compounding, such as the active compound for the treatment of hypersensitivity in an individual. Determined by and directly dependent on inherent limits.

The antibody molecules are about 35 mg/m ² to about 440 mg/m ² , typically about 70 mg/m ² to about 310 mg/m ² , and more typically about 110 mg/m ² to about 130 mg/m ² may be administered by intravenous infusion at a rate of greater than 20 mg/min, such as 20-40 mg/min, and typically 40 mg/min or more, to reach a dose of . In embodiments, a level of about 3 mg/kg is achieved with an infusion rate of about 110 mg/m ² to about 130 mg/m ² . In other embodiments, the antibody molecules are about 1 mg/m ² to about 100 mg/m ² , such as about 5 mg/m ² to about 50 mg/m ² , about 7 mg/m ² to about 25 mg/m ² , or It may be administered by intravenous infusion at a rate of less than 10 mg/min, such as 5 mg/min or less, to reach a dose of about 10 mg/ ^m2 . In some embodiments, the antibody is infused over about 30 minutes. It should be noted that dosage values may vary depending on the type and severity of the condition sought to be alleviated. Furthermore, for any particular subject, specific dosage regimens will be adjusted over time based on individual needs and the professional judgment of the person administering the composition or supervising its administration. It should be understood that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. "Therapeutically effective amount" refers to an amount effective, at dosages and for times necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a modified antibody or antibody fragment may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment being modified are outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably increases a measurable parameter, e.g., tumor growth rate, by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%. Measurable parameters of a compound, such as the ability to inhibit cancer, can be evaluated in animal model systems that are predictive of efficacy in human tumors. Alternatively, this property of a composition may be assessed by examining the ability of the compound to inhibit such inhibition in vitro by assays known to those skilled in the art.

A "prophylactically effective amount" refers to an amount effective, at dosages and for the times necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose will be used in a subject before the disease or in the early stages of the disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

Also within the scope of this disclosure are kits containing the combinations, compositions, or formulations described herein. The kit includes instructions for use (e.g., following the dosage regimens described herein); other reagents, e.g., a label, a therapeutic agent, or chelating an antibody to a label or therapeutic agent; or other agents useful for coupling, or radioprotective compositions; devices or other materials for preparing antibodies for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to subjects. It may include one or more other elements, including other materials.

Chemotherapeutic Agents The compounds disclosed throughout, such as TGFβ inhibitors (eg, NIS793) or PD-1 inhibitors (eg, tislelizumab), can be used in conjunction with chemotherapeutic agents. Therapeutic agents include, but are not limited to, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection. (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), Chlorambucil (Leukeran®), Cisplatin (Platinol®), Cladribine (Leustatin®), Cyclophosphamide (Cytoxan® or Neosar®), Cytarabine, Cytosine Arabino (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine ( Daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid) (R)), fludarabine phosphate (Fludara(R)), 5-fluorouracil (Adrucil(R), Efudex(R)), flutamide (Eulexin(R)), tezacytibine (Tezacytibine), gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®). ), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Prinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), Mylotarg , paclitaxel (Taxol®) or nab-paclitaxel, Fenix (Yttrium90/MX-DTPA), pentostatin, polyfeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex(R)) ), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®) ), vincristine (Oncovin®), vinorelbine (Navelbine®), epirubicin (Elence®), oxaliplatin (Eloxatin®), exemestane (Aromasin®), letrozole (Femara®), and fulvestrant (Faslodex®).

In some embodiments, gemcitabine can be used in conjunction with any of the therapeutic molecules disclosed throughout. For example, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with gemcitabine to treat a patient. A PD-1 inhibitor (eg, PDR001, BGB-A317, or BGB-108) can be used in combination with gemcitabine or gemcitabine and a TGFβ inhibitor.

In some embodiments, nab-paclitaxel can be used in conjunction with any of the therapeutic molecules disclosed throughout. For example, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with nab-paclitaxel to treat a patient. A PD-1 inhibitor (eg, PDR001, BGB-A317, or BGB-108) can be used in combination with nab-paclitaxel or nab-paclitaxel and a TGFβ inhibitor (eg, NIS793). In some combinations, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with gemcitabine and nab-paclitaxel. In some combinations, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with a PD-1 inhibitor (eg, PDR001, BGB-A317, or BGB-108), gemcitabine, and nab-paclitaxel.

When used in conjunction with other therapeutic agents, gemcitabine may be administered intravenously to the patient. For example, gemcitabine can be administered intravenously to a patient at a dose of 1000 mg/ ^m2 . Gemcitabine may be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, gemcitabine is administered at a dose of 1000 mg/ ^m2 on days 1, 8, and 15 of each cycle (eg, a 21-day or 38-day cycle). In some instances, gemcitabine is administered intravenously to the patient at a dose of 675 mg/ ^m2 . For example, gemcitabine is administered to patients at a dose of 675 mg/m ² on days 1 and 8 of each cycle (eg, a 21-day or 28-day cycle).

When nab-paclitaxel is used in conjunction with other therapeutic agents, it may be administered intravenously to the patient. For example, nab-paclitaxel can be administered intravenously to a patient at a dose of 125 mg/m ² . Nab-paclitaxel may also be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, nab-paclitaxel is administered at a dose of 125 mg/m ² on days 1, 8, and 15 of each cycle (eg, a 21-day or 28-day cycle).

In some embodiments, 5-fluorouracil can be used in conjunction with any of the therapeutic molecules disclosed throughout. For example, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with 5-fluorouracil to treat a patient.

When 5-fluorouracil is used in conjunction with other therapeutic agents, it may be administered intravenously to the patient. For example, 5-fluorouracil may be administered to a patient intravenously (eg, using an intravenous bolus) at a dose of 400-2400 mg/m ² . 5-fluorouracil may also be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, 5-fluorouracil is administered as an intravenous bolus at 400 mg/ ^m2 on days 1 and 15 of each cycle (e.g., a 21-day or 28-day cycle), followed by a continuous intravenous infusion for 46 hours. Administered at 2400 mg/ ^m2 .

In some embodiments, leucovorin can be used in conjunction with any of the therapeutic molecules disclosed throughout. For example, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with leucovorin to treat a patient.

When leucovorin is used in conjunction with other therapeutic agents, it may be administered intravenously to the patient. For example, leucovorin can be administered intravenously to a patient at a dose of 400 mg/ ^m2 . Leucovorin may also be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, leucovorin is administered at a dose of 400 mg/m ² on days 1 and 15 of each cycle (eg, a 21-day or 28-day cycle).

In some embodiments, levoleucovorin can be used as a replacement for leucovorin. In those cases, levoleucovorin may be administered at 200 mg/ ^m2 . Additionally, levoleucovorin may be administered on days 1 and 15 of a 28 day cycle.

In some embodiments, oxaliplatin can be used in conjunction with any of the therapeutic molecules disclosed throughout. For example, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with oxaliplatin to treat a patient.

When oxaliplatin is used in combination with other therapeutic agents, it may be administered intravenously to a patient. For example, oxaliplatin may be administered intravenously to a patient at a dose of 85 mg/ ^m2 . Oxaliplatin may also be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, oxaliplatin may be administered at a dose of 85 mg/ ^m2 on days 1 and 15 of each cycle (e.g., a 21-day or 28-day cycle).

In some embodiments, irinotecan can be used in conjunction with any of the therapeutic molecules disclosed throughout. For example, a TGFβ inhibitor (eg, NIS793) can be used in conjunction with irinotecan to treat a patient.

When irinotecan is used in conjunction with other therapeutic agents, it may be administered intravenously to the patient. For example, irinotecan may be administered intravenously to a patient at a dose of 180 mg/ ^m2 . Irinotecan may also be administered once a week, once every two weeks, once every three weeks, or once every four weeks for a given period of time. For example, irinotecan is administered at a dose of 180 mg/m ² on days 1 and 15 of each cycle (eg, a 21-day or 28-day cycle).

In some embodiments, a TGFβ inhibitor (eg, NIS793) can be combined with gemcitabine and nab-paclitaxel. In some embodiments, a TGFβ inhibitor (eg, NIS793) can be combined with a PD-1 inhibitor (eg, PDR001, BGB-A317, or BGB-108), gemcitabine, and nab-paclitaxel. In some embodiments, a TGFβ inhibitor (eg, NIS793) can be combined with bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin. In some embodiments, the TGFβ inhibitor (e.g., NIS793) is combined with a PD-1 inhibitor (e.g., PDR001, BGB-A317, or BGB-108), bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin. can be In some embodiments, a TGFβ inhibitor (eg, NIS793) can be combined with bevacizumab, 5-fluorouracil, leucovorin, and irinotecan. In some embodiments, a TGFβ inhibitor (e.g., NIS793) is combined with a PD-1 inhibitor (e.g., PDR001, BGB-A317, or BGB-108), bevacizumab, 5-fluorouracil, leucovorin, and irinotecan. obtain.

In some embodiments, cyclophosphamide can be used in conjunction with any of the therapeutic molecules disclosed throughout, such as a TGFβ inhibitor (eg, NIS793), to treat a patient. When cyclophosphamide is used in conjunction with other therapeutic agents, such as TGFβ inhibitors (eg, NIS793), it may be administered intravenously or orally to the patient. For example, cyclophosphamide can be administered intravenously to a patient at a dose of 250 mg/ ^m2 . Cyclophosphamide may also be administered daily for a period of time. For example, cyclophosphamide may be administered for 5 days, such as 5 consecutive days. For example, cyclophosphamide is administered at a dose of 250 mg/m ² for 5 consecutive days.

In some embodiments, topotecan can be used in conjunction with any of the therapeutic molecules disclosed throughout, such as a TGFβ inhibitor (eg, NIS793), to treat a patient. When topotecan is used in conjunction with other therapeutic agents, such as a TGFβ inhibitor (eg, NIS793), it may be administered intravenously to the patient. For example, topotecan may be administered intravenously to a patient at a dose of 0.75 mg/ ^m2 . Topotecan may also be administered over a period of time, such as 30 minutes. Topotecan may also be administered for 5 days, such as 5 consecutive days. For example, topotecan is administered at a dose of 0.75 mg/ ^m2 for 5 consecutive days.

Additional Exemplary Combinations and Usage The compounds and/or therapeutic agents disclosed throughout can be used in any combination. Additionally, each therapeutic agent can be used in a manner that is not unduly toxic, yet effective for its intended purpose. The following combinations and/or uses are presented for illustrative purposes only and are not exhaustive of all contemplated combinations and/or uses. Those skilled in the art can further envision various combinations and/or uses disclosed throughout this specification.

In one combination, NIS793 is administered to a patient along with gemcitabine and nab-paclitaxel. The three combinations are administered intravenously to the patient. NIS793 is administered to patients in this triple combination at a dose of 2100 mg/m ² on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). Gemcitabine is administered to patients in this triple combination at a dose of 1000 mg/m ² on days 1, 8, and 15 of each cycle (eg, 21-day or 28-day cycle). Nab-paclitaxel is administered to patients in this triple combination at a dose of 125 mg/m ² on days 1, 8, and 15 of each cycle (eg, 21-day or 28-day cycle). This combination treats pancreatic cancer, such as metastatic pancreatic adenocarcinoma.

In one combination, NIS793 is administered to a patient with bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin. The four combinations are administered to the patient intravenously (possibly as an intravenous bolus or continuous intravenous infusion). NIS793 is administered to patients in this quadruple combination at a dose of 2100 mg/m ² on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). Bevacizumab is administered to patients in this quadruple combination at a dose of 5 mg/kg on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). 5-Fluorouracil is administered to patients as an intravenous bolus at a dose of 400 mg/ ^m2 on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycles), followed by a continuous intravenous infusion for 46 hours. administered at 2400 mg/ ^m2 . Leucovorin is administered to patients in this quadruple combination at a dose of 400 mg on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). Oxaliplatin is administered to patients in this quadruple combination at a dose of 85 mg/m ² on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). This combination treats colorectal cancer, such as metastatic colorectal cancer.

In one combination, NIS793 is administered to a patient with bevacizumab, 5-fluorouracil, leucovorin, and irinotecan. The five combinations are administered to the patient intravenously (possibly as an intravenous bolus or continuous intravenous infusion). NIS793 is administered to patients in this five-way combination at a dose of 2100 mg on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). Bevacizumab is administered to patients in this five-way combination at a dose of 5 mg/kg on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). 5-Fluorouracil is administered as an intravenous bolus at a dose of 400 mg/ ^m2 to patients on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycles) in this quintuple combination, followed by Administered at 2400 mg/m ² by continuous intravenous infusion for 46 hours. Leucovorin is administered to patients in this five-way combination at a dose of 400 mg/m ² on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). Irinotecan is administered to patients in this five-way combination at a dose of 180 mg/m ² on days 1 and 15 of each cycle (eg, 21-day or 28-day cycle). This combination treats colorectal cancer, eg, metastatic colorectal cancer.

In certain combinations, NIS793 is administered to a patient along with oxaliplatin and capecitabine. The three-way combination is administered to the patient intravenously (possibly as an intravenous bolus or continuous intravenous infusion). NIS793 will be administered to patients once every three weeks (Q3W) at a dose of 2100 mg in this triple combination. Oxaliplatin is administered intravenously to patients in this triple combination cycle on day 1 of the Q3W cycle at a dose of 130 mg/ ^m2 . Capecitabine will be administered to patients in this triple combination twice daily (days 1-14) at a dose of 1000 mg/m ² in Q3W cycles. Tislelizumab is optionally administered to patients at a once every three weeks (Q3W) dose of 200 mg. With this combination, gastric cancer is treated.

In one combination, NIS793 is administered to a patient along with oxaliplatin, leucovorin (or levoleucovorin) and 5-fluorouracil. The combination is administered to the patient intravenously (possibly as an intravenous bolus or continuous intravenous infusion). NIS793 will be administered to patients in this quadruple combination once every three weeks (Q3W) at a dose of 2100 mg. Oxaliplatin will be administered to patients on day 1 of the Q2W cycle at a dose of 85 mg/ ^m2 . Leucovorin is administered to the patient on day 1 of the Q2W cycle at a dose of 400 mg/m ² (or levoleucovorin is administered at a dose of 200 mg/m ² ). 5-Fluorouracil is administered intravenously at 400 mg/m ² on day 1 of the Q2W cycle, followed by 1200 mg/m ² on days 1-2. Tislelizumab is optionally administered once every four weeks (Q4W) at a dose of 300 mg. With this combination, gastric cancer is treated.

In one combination, NIS793 is administered to a patient with oxaliplatin and capecitabine. The combination is administered to the patient intravenously (possibly as an intravenous bolus or continuous intravenous infusion). NIS793 will be administered to patients in this triple combination once every two weeks (Q2W) at a dose of 2100 mg. Oxaliplatin will be administered to patients intravenously in this triple combination on day 1 of the Q3W cycle at a dose of 130 mg/ ^m2 . Capecitabine is administered orally twice daily (days 1-14) at a dose of 1000 mg/m ² in Q3W cycles in this triple combination. Tislelizumab will be administered once every three weeks (Q3W) at a dose of 200 mg. With this combination, gastric cancer is treated.

In one combination, NIS793 is administered to a patient along with oxaliplatin, leucovorin (or levoleucovorin) and 5-fluorouracil. The combination is administered to the patient intravenously (possibly as an intravenous bolus or continuous intravenous infusion). NIS793 will be administered to patients in this quadruple combination once every two weeks (Q2W) at a dose of 2100 mg. Oxaliplatin will be administered to patients on day 1 of the Q2W cycle at a dose of 85 mg/ ^m2 . Leucovorin is administered to the patient on day 1 of the Q2W cycle at a dose of 400 mg/m ² (or levoleucovorin is administered at a dose of 200 mg/m ² ). 5-Fluorouracil is administered intravenously at 400 mg/m ² on day 1 of the Q2W cycle, followed by 1200 mg/m ² on days 1-2. Tislelizumab is optionally administered once every four weeks (Q4W) at a dose of 300 mg. With this combination, gastric cancer is treated.

In certain combinations, NIS793 is administered to a patient with cyclophosphamide or topotecan. This three-way combination is administered intravenously to the patient. NIS793 is administered in this triple combination based on patient weight: (a) at a dose of 45 mg/kg for patients weighing less than 20 kg; (b) at a dose of 30 mg/kg for patients weighing 20-40 kg. and (c) at a dose of 20 mg/kg for patients weighing more than 40 kg. Cyclophosphamide is administered intravenously to patients in this triple combination at a dose of 250 mg/m ² for 5 consecutive days (days 1-5). Topotecan is administered intravenously to patients in this triple combination at a dose of 0.75 mg/m ² for 5 consecutive days (days 1-5). In some cases, this triple combination can be used to treat neuroblastoma. In some cases, the patient population is a pediatric patient population (eg, under 18 years of age).

In certain combinations, NIS793 is administered to a patient along with gemcitabine. This combination is administered intravenously to the patient. NIS793 is administered in this combination based on patient weight: (a) at a dose of 45 mg/kg for patients weighing less than 20 kg; (b) at a dose of 30 mg/kg for patients weighing 20-40 kg. and (c) at a dose of 20 mg/kg for patients weighing more than 40 kg; Gemcitabine is administered to the patient intravenously in this combination once a week, for example on days 1 and 8 at a dose of 675 mg/m ² . In some cases, this combination can be used to treat osteosarcoma. In some cases, the patient population is a pediatric patient population (eg, under 18 years of age).

Diagnosis of Subjects and Treatment of Subjects As used herein, the term "subject" is intended to include humans and non-human animals. In some embodiments, the subject is a human subject. The term "non-human animal" includes mammals and non-mammals, such as non-human primates. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient in need of an enhanced immune response. The combinations described herein are suitable for the treatment of human patients with disorders that can be treated by modulating (eg, increasing or inhibiting) the immune response. In certain embodiments, the patient has or is at risk of having a disorder described herein, such as a cancer described herein.

In some cases, a subject being treated using the methods disclosed herein knows for themselves that they have a disease or condition that would, in some cases, benefit from the methods described herein. For example, in some cases, the subject has been tested and/or diagnosed for a disease. This testing and/or diagnosis may be made by a physician or other qualified medical professional. In some cases, the testing and/or diagnosis may be self-performed based on one or more symptoms, such as a bulging mass, a lump, etc. Thus, in some embodiments, the subject may be in need of the methods described herein to treat the disease or condition. The term "in need of" is intended to describe that the subject (or the person treating the subject) knows of the existence of a condition or disease (e.g., a proliferative disease such as cancer).

In certain embodiments, the subject has been identified as having TGFβ (1, 2, or 3) expression in its one or more tumors (or tumor microenvironment). In certain embodiments, the subject has been identified as having PD-1 expression in its one or more tumors (or tumor microenvironment). In certain embodiments, the subject has been identified as having PD-L1 expression in its one or more tumors (or tumor microenvironment). In certain embodiments, the subject has been identified as having PD-L2 expression in its one or more tumors (or tumor microenvironment). In some embodiments, the subject has been identified as having expression of both TGFβ (1, 2, or 3) and PD-1 in its one or more tumors (or tumor microenvironment). In some embodiments, the subject has been identified as having expression of both TGFβ (1, 2, or 3) and PD-L1 in its one or more tumors (or tumor microenvironment). In some embodiments, the subject has been identified as having expression of both TGFβ (1, 2, or 3) and PD-L2 in its one or more tumors (or tumor microenvironment). Once these biomarkers are found, treatment using the methods described can then be used.

In some embodiments, the subject is about 5 kg to about 500 kg. In some embodiments, the subject is about 10 kg to about 400 kg. In some embodiments, the subject is about 15 kg to about 300 kg. In some embodiments, the subject is about 20 kg to about 200 kg. In some embodiments, the subject is about 25 kg to about 150 kg. In some embodiments, the subject is about 40 kg to about 125 kg. In some embodiments, the subject is about 50 kg to about 100 kg. In some embodiments, the subject is about 65 kg to about 85 kg. In some embodiments, the subject is about 40 kg. In some embodiments, the subject is about 45 kg. In some embodiments, the subject is about 50 kg. In some embodiments, the subject is about 55 kg. In some embodiments, the subject is about 60 kg. In some embodiments, the subject is about 65 kg. In some embodiments, the subject is about 70 kg. In some embodiments, the subject is about 75 kg. In some embodiments, the subject is about 80 kg. In some embodiments, the subject is about 85 kg. In some embodiments, the subject is about 90 kg. In some embodiments, the subject is about 95 kg. In some embodiments, the subject is about 100 kg. In some embodiments, the subject is about 110 kg. In some embodiments, the subject is about 120 kg. In some embodiments, the subject is about 130 kg. In some embodiments, the subject is about 140 kg. In some embodiments, the subject is about 150 kg.

Cancer In some embodiments, the method provides a method for treating myelofibrosis (e.g., primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (PET-MF), post-polycythemia vera myelofibrosis). (PPV-MF)), leukemia (e.g., acute myeloid leukemia (AML), e.g., relapsed or refractory AML or de novo AML; or chronic lymphocytic leukemia (CLL)), lymphoma (e.g., T-cell lymphoma, B-cell lymphoma, non-Hodgkin lymphoma, or small lymphocytic lymphoma (SLL)), myeloma (e.g., multiple myeloma), lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., squamous and/or NSCLC with squamous histology or NSCLC adenocarcinoma), or small cell lung cancer (SCLC)), skin cancer (e.g. Merkel cell carcinoma or melanoma (e.g. advanced melanoma)), ovarian cancer, mesothelioma , bladder cancer, soft tissue sarcoma (e.g., hemangiopericytoma (HPC)), bone cancer (osteosarcoma), kidney cancer (e.g., renal cancer (e.g., renal cell carcinoma)), liver cancer (e.g., hepatocellular carcinoma) , cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS) (e.g., low-risk MDS), prostate cancer, breast cancer (e.g., expressing one, two, or all of estrogen receptors, progesterone receptors, or Her2/neu) breast cancer (e.g., triple-negative breast cancer), colorectal cancer, nasopharyngeal cancer, duodenal cancer, endometrial cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)), head and neck cancer (e.g., head and neck squamous cell carcinoma) (HNSCC), anal cancer, gastroesophageal cancer, thyroid cancer (eg, undifferentiated thyroid cancer), cervical cancer, or neuroendocrine tumor (NET) (eg, atypical pulmonary carcinoid tumor).

In certain embodiments, the patient is not suitable for standard treatment regimens with established benefits in patients with one or more of the cancers described herein. In some embodiments, the subject is unsuitable for chemotherapy. In some embodiments, the chemotherapy is intensive induction chemotherapy. For example, the methods described herein can be used to treat adult patients with one or more of the cancers as described. In certain embodiments, the inhibitor (TGFβ and/or PD1) is administered in an amount effective to treat cancer or a condition thereof.

The compositions, formulations, or methods described herein can be used to inhibit the growth of cancerous tumors. Alternatively, the compositions, formulations, or methods described herein may be used in conjunction with standard-of-care treatments for cancer, another antibody or antigen-binding fragment thereof, an immunomodulatory agent (e.g., a co-administrator), as described herein. activators of stimulating molecules or inhibitors of inhibitory molecules); vaccines, such as therapeutic cancer vaccines; or other forms of cellular immunotherapy. In one embodiment, the method is suitable for treating cancer in vivo.

In another embodiment, a method of treating a subject, e.g., reducing or ameliorating a hyperproliferative condition or disorder (e.g., cancer), e.g., a solid tumor, hematologic cancer, soft tissue tumor, or metastatic lesion, in a subject is provided. provided. The method includes practicing a method described herein in accordance with the dosage regimen disclosed herein, or a composition or formulation described herein in accordance therewith.

As used herein, the term "cancer" refers to any type of cancerous growth or carcinogenic process, metastatic tissue or malignantly transformed cells, regardless of histopathological type or stage of invasiveness; It is intended to include tissues or organs. Examples of cancerous disorders include, but are not limited to, hematological cancers, solid tumors, soft tissue tumors, and metastatic lesions.

Examples of solid tumors include, but are not limited to, liver, lung, breast, lymphatic system, gastrointestinal (e.g., colon), anus, genital and genitourinary organs (e.g., kidney, urothelium, bladder), prostate, CNS ( Malignant tumors of various organ systems, such as those that occur in the brain, nervous system or glial cells), head and neck, skin, pancreas, and pharynx, such as sarcomas, and carcinomas (including adenocarcinomas and squamous cell carcinomas) can be mentioned. Adenocarcinomas include most colon cancers, rectal cancers, renal cell carcinomas (e.g., clear cell or non-clear cell renal cell carcinomas), liver cancers, lung cancers (e.g., non-lung small cell carcinomas (e.g. , squamous or non-squamous non-small cell lung cancer), small intestine cancer, and esophageal cancer. Squamous cell cancers include, for example, lung, esophagus, skin, head and neck region, oral cavity, anus, and cervical malignancies. In one embodiment, the cancer is melanoma, eg, advanced stage melanoma. The cancer may be early stage, intermediate stage, late stage or metastatic cancer. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the combinations described herein.

In certain embodiments, the cancer is a solid tumor. In some embodiments, the cancer is ovarian cancer. In other embodiments, the cancer is lung cancer, such as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In other embodiments, the cancer is mesothelioma. In other embodiments, the cancer is a skin cancer, such as Merkel cell carcinoma or melanoma. In other embodiments, the cancer is renal cancer, eg, renal cell carcinoma (RCC). In other embodiments, the cancer is bladder cancer. In other embodiments, the cancer is a soft tissue sarcoma, such as a hemangiopericytoma (HPC). In other embodiments, the cancer is bone cancer, eg, osteosarcoma. In other embodiments, the cancer is colorectal cancer. In other embodiments, the cancer is pancreatic cancer (eg, PDAC). In other embodiments, the cancer is nasopharyngeal carcinoma. In other embodiments, the cancer is breast cancer. In other embodiments, the cancer is duodenal cancer. In other embodiments, the cancer is endometrial cancer. In other embodiments, the cancer is an adenocarcinoma, eg, an adenocarcinoma of unknown primary. In other embodiments, the cancer is liver cancer, eg, hepatocellular carcinoma. In other embodiments, the cancer is cholangiocarcinoma. In other embodiments, the cancer is a sarcoma. In certain embodiments, the cancer is a myelodysplastic syndrome (MDS) (eg, high-risk MDS or low-risk MDS). In some embodiments, the cancer is neuroblastoma. In certain embodiments, the cancer is osteosarcoma.

In another embodiment, the cancer is carcinoma (eg, advanced or metastatic cancer), melanoma, or lung cancer, eg, non-small cell lung cancer. In one embodiment, the cancer is lung cancer, eg, non-small cell lung cancer or small cell lung cancer. In some embodiments, the non-small cell lung cancer is stage I (e.g., stage Ia or Ib), stage II (e.g., stage IIa or IIb), stage III (e.g., stage IIIa or IIIb), or stage IV non- It is small cell lung cancer. In one embodiment, the cancer is melanoma, eg, advanced melanoma. In one embodiment, the cancer is an advanced or unresectable melanoma that is refractory to other therapies. In other embodiments, the cancer is melanoma with a BRAF mutation (eg, a BRAF V600 mutation). In another embodiment, the cancer is hepatocellular carcinoma, eg, advanced hepatocellular carcinoma, with or without viral infection, eg, chronic viral hepatitis. In another embodiment, the cancer is prostate cancer, eg, advanced prostate cancer. In yet another embodiment, the cancer is myeloma, eg, multiple myeloma. In yet another embodiment, the cancer is renal cancer, e.g., renal cell carcinoma (RCC) (e.g., metastatic RCC, non-clear cell renal cell carcinoma (nccRCC), or clear cell renal cell carcinoma (CCRCC)). It is.

In some embodiments, the cancer is an MSI-high cancer. In some embodiments, the cancer is metastatic cancer. In other embodiments, the cancer is an advanced cancer. In other embodiments, the cancer is a recurrent or refractory cancer.

Exemplary cancers whose growth may be inhibited using the methods, compositions, or formulations as disclosed herein include cancers that typically respond to immunotherapy. Additionally, the combinations described herein can be used to treat refractory or recurrent malignancies.

Examples of other cancers that may be treated include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain/CNS cancer; primary CNS lymphoma; central nervous system (CNS) neoplasm; breast cancer; Cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; intraepithelial neoplasm; renal cancer; laryngeal cancer; leukemia ( acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic or acute leukemia); liver cancer; lung cancer (e.g. small cell and non-small cell); Hodgkin and non-Hodgkin lymphoma lymphomas, including; lymphocytic lymphoma; melanoma, such as cutaneous or intraocular malignant melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer ; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system cancer, hepatocellular carcinoma, anal cancer, Fallopian tube cancer, vaginal cancer, vulvar cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, spinal axis tumor, brainstem glioma, pituitary adenoma , Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, and other carcinomas and sarcomas, and combinations of the foregoing cancers.

The methods and therapies described herein include one or more therapeutic agents, such as one or more anti-cancer agents, cytotoxic or cytostatic agents, hormonal treatments, vaccines, and/or other immunotherapies. may include compositions that are co-formulated with and/or co-administered with. In other embodiments, the antibody molecules are administered in combination with other therapeutic treatment modalities including surgery, radiation, cryosurgery, and/or hyperthermia. Such combination therapy advantageously utilizes lower dosages of the therapeutic agents administered, thereby avoiding the potential toxicity or complications associated with various monotherapies.

When administered in combination, the TGF-β inhibitor, PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor, one or more additional agents, or all may be administered individually, e.g. as a single agent. It can be administered in higher, lower, or the same amount or dosage as compared to the amount or dosage of each drug used as therapy. In certain embodiments, the TGF-β inhibitor, PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor, one or more additional agents, or all administered amounts or dosages are , individually, eg, at least 20%, at least 30%, at least 40%, or at least 50% lower than the amount or dosage of each agent used as monotherapy. In other embodiments, a TGF-β inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, or a PD-L2 inhibitor, one or more additional agents, or all of the desired effects (e.g., cancer (eg, at least 20%, at least 30%, at least 40%, or at least 50% lower).

In other embodiments, the additional therapeutic agent is from the agents listed in Table 6 of WO 2017/019897. In some embodiments, the additional therapeutic agent is 1) a protein kinase C (PKC) inhibitor; 2) a heat shock protein, e.g., as described in Table 6 of WO 2017/019897; 90 (HSP90) inhibitors; 3) inhibitors of phosphoinositide 3-kinase (PI3K) and/or target of rapamycin protein (mTOR); 4) inhibitors of cytochrome P450 (e.g., CYP17 inhibitors or 17α-hydroxylase/C17- 20 lyase inhibitors); 5) iron chelators; 6) aromatase inhibitors; 7) inhibitors of p53, e.g., inhibitors of p53/Mdm2 interaction; 8) apoptosis inducers; 9) angiogenesis inhibitors; 10 ) Aldosterone synthase inhibitors; 11) Smoothened (SMO) receptor inhibitors; 12) Prolactin receptor (PRLR) inhibitors; 13) Wnt signaling inhibitors; 14) CDK4/6 inhibitors; 15) Fibroblasts Growth factor receptor 2 (FGFR2)/fibroblast growth factor receptor 4 (FGFR4) inhibitors; 16) Inhibitors of macrophage colony stimulating factor (M-CSF); 17) c-KIT, histamine release, Flt3 (e.g. , FLK2/STK1) or PKC; 18) an inhibitor of one or more of VEGFR-2 (e.g. FLK-1/KDR), PDGFRβ, c-KIT or Raf Kinase C; ;19) somatostatin agonists and/or growth hormone release inhibitors;20) anaplastic lymphoma kinase (ALK) inhibitors;21) insulin-like growth factor 1 receptor (IGF-1R) inhibitors;22) P-glycoprotein 1 inhibitors; 23) vascular endothelial growth factor receptor (VEGFR) inhibitors; 24) BCR-ABL kinase inhibitors; 25) FGFR inhibitors; 26) inhibitors of CYP11B2; 27) HDM2 inhibitors, such as HDM2-p53 Inhibitors of interactions; 28) Inhibitors of tyrosine kinases; 29) Inhibitors of c-MET; 30) Inhibitors of JAK; 31) Inhibitors of DAC; 32) Inhibitors of 11β-hydroxylase; 33) IAPs 34) Inhibitors of PIM kinases; 35) Inhibitors of Porcupine; 36) Inhibitors of BRAF, such as BRAF V600E or wild type BRAF; 37) Inhibitors of HER3; 38) Inhibition of MEK or 39) one or more of the following: drugs; or 39) inhibitors of lipid kinases.

Pediatric Patients In some cases, the patient population can be an adult population or a pediatric population. For pediatric patients, NIS793 can be used to treat children with relapsed or refractory solid tumors. Monotherapy and/or combination therapy can be used to treat relapsed or refractory solid tumors.

In some embodiments, NIS793 (or other TGFβ inhibitors), cyclophosphamide and topotecan are used to treat patients, such as pediatric patients, with neuroblastoma, such as relapsed or refractory neuroblastoma. can be treated.

In some embodiments, NIS793 (or other TGFβ inhibitors) and gemcitabine can be used to treat patients, such as pediatric patients, with osteosarcoma, including recurrent measurable osteosarcoma.

In some embodiments, the patient population is more than 12 months old and less than 21 years old. For example, when treating neuroblastoma, the patient population may be over 12 months of age and under 21 years of age. In some embodiments, the patient population includes adult patients who can be 12 months or older and up to 39 years of age (12 months or older and 39 years or younger).

Patients treated with the various therapies (and combinations) described throughout must have histological verification of malignancy at initial diagnosis or recurrence. Patients with relapsed or refractory solid tumors can be treated with a variety of therapies (and combinations) described throughout. In some embodiments, patients with relapsed or refractory neuroblastoma may be treated with various therapies (and combinations) described throughout. In some embodiments, patients with relapsed or refractory osteosarcoma may be treated with various therapies (and combinations) described throughout.

In some embodiments, a TGFβ inhibitor (eg, NIS793) can be administered to a patient at a dose of 45 mg/kg for patients weighing less than 20 kg. In some embodiments, a TGFβ inhibitor (eg, NIS793) can be administered to a patient at a dose of 30 mg/kg for a patient weighing 20-40 kg. In some embodiments, a TGFβ inhibitor (eg, NIS793) can be administered to a patient at a dose of 20 mg/kg for patients weighing more than 40 kg. Administration of each group can be once every three weeks, which is considered a single cycle. In some cases, patients may be treated for up to 35 cycles.

For some patients (e.g., those with neuroblastoma and therefore "in need"), cyclophosphamide and/or topotecan may be administered in conjunction with a TGFβ inhibitor (e.g., NIS793). can be done. For some patients (eg, those suffering from osteosarcoma and thus "in need"), gemcitabine may be administered in conjunction with a TGFβ inhibitor (eg, NIS793).

In some embodiments, TGFβ inhibitors (eg, NIS793) may be used in conjunction with additional therapeutic agents. In some cases, additional therapeutic agents include cyclophosphamide or topotecan. If cyclophosphamide is used, it may be administered at 250 mg/ ^m2 . In some embodiments, cyclophosphamide is administered for 5 days, such as 5 consecutive days. In some embodiments, the additional therapeutic agent comprises topotecan administered at 0.75 mg/ ^m2 . If topotecan is used, it may be administered for 5 days, for example 5 consecutive days. In some embodiments, the proliferative disease is neuroblastoma.

In some embodiments, the additional therapeutic agent includes gemcitabine. If gemcitabine is used, it may be administered at 675 mg/ ^m2 . In some embodiments, gemcitabine is administered for two days, such as days 1 and 8. In some embodiments, the proliferative disease is osteosarcoma.

In some examples, each cycle is a 21 day period. Patients may be treated for approximately 2 years.

Example 1: Drug Product A TGFβ inhibitor known as NIS793 was made into a powder that could be used as a solution for injection. The powder was provided in a glass vial with a rubber stopper, which was sealed with a flip-off cap. Each vial contained 100 mg of NIS793 lyophilizate. This drug product was manufactured using standard aseptic processing techniques. This drug product contained, in addition to NIS 793, the following pharmaceutical excipients: L-histidine/L-histidine hydrochloride monohydrate, polysorbate 20 and sucrose. Vials were provided with 20% overfill to allow for aspiration of the entire dose.

This drug product was designed to be reconstituted with 1 mL of sterile water for injection prior to administration to yield a 100 mg/mL NIS793 solution.

NIS793 concentrate for injection solution was provided in glass vials with rubber stoppers, which were sealed with flip-off caps. Each vial contained 700 mg of NIS793 in 7 mL of solution. This drug product solution contained the same amount and quality of excipients as the NIS 793 powder for injection solution after reconstitution with sterile water. Similarly, it was provided with a 7% overfill to allow for aspiration of the entire dose.

Example 2: Human Study A drug product containing NIS793 as described in Example 1 was used in a clinical study. A summary of ongoing human trials is provided in Table 5 below.

One human trial has been initiated and is ongoing: First in Human Study, CNIS793X2101, "Phase I/Ib, Open-Label Study of NIS793 in Combination with PDR001 in Adult Patients with Advanced Malignancies" , a multicenter dose-escalation study.” A total of 120 patients were treated with NIS793 as a single agent or in combination with PDR001 (Table 5).

Pharmacokinetics, Metabolism and Pharmacodynamics in Humans PK data for NIS793 from the CNIS793X2101 study (75 patients, cut-off date May 4, 2020) was characterized. For NIS793 as a single agent (NIS793: 0.3-1 mg/kg Q3W) and in combination with PDR001 (NIS793/PDR001: 0.3 mg/kg/100 mg Q3W, 0.3-30 mg/kg/300 mg Q3W and 20 A summary of the derived PK parameter estimates in the dose escalation cohort at ~30 mg/kg Q2W/400 mg Q4W) is provided in Table 6 for cycle 1 and in Table 7 for cycle 3. In addition, a summary of the derived PK parameter estimates in the dose expansion cohort in combination with PDR001 (NIS793/PDR001: 2100 mg/300 mg Q3W) for NIS793 in MSS-CRC and NSCLC is provided in Table 8. The average concentration-time profile for each dose cohort of NIS793 is plotted in FIG. 1 for cycle 1 and in FIG. 2 for cycle 3.

After administration of NIS793 by 30 minute intravenous infusion, an approximately dose-proportional increase in NIS793 exposure (ie, Cycle 1 Cmax and AUClast) was observed from 0.3 mg/kg to 30 mg/kg. Based on the ratio of AUClast and Cmax comparing cycle 3 to cycle 1, a moderate accumulation of NIS793 (up to approximately 2.0-fold) was observed. PK variation was low to moderate (CV%) as indicated by inter-subject variation (eg 12.1-73.3% for Cmax).

In combination with NIS793, PDR001 was administered in three doses and two dosing regimens (100 or 300 mg Q3W and 400 mg Q4W). The PK of PDR001 in combination with NIS793 appears similar to single agent data from the PDR001 clinical trial study.

Population PK analysis on concentration data from the dose escalation phase of study CNIS793X2101 was used to describe the pharmacokinetic properties of NIS793, including the effect of body weight as a covariate on clearance and volume of distribution. This analysis suggested that the pharmacokinetics of NIS793 can be well described using a two-compartment model that includes first-order elimination from the central compartment. This is consistent with the observation that NIS793 PK appears to be dose-proportional and time-independent based on non-compartmental analysis.

Body weight (BW) is a covariate for clearance in the population PK model with an estimated exponent of 0.55 (CV% = 40%) from the power model, but the expected difference between weight-based and fixed-dose regimens is Exposure and steady-state trough concentrations were comparable between different BW categories. This analysis supports the use of a constant or fixed dose regimen on a mg basis, independent of patient weight, as weight-based dosing regimens do not reduce interindividual variability. Model-based simulations indicated that a dose of 2100 mg would correspond to the exposure observed at 30 mg/kg. Furthermore, a dose of 1400 mg would correspond to the exposure observed at 20 mg/kg.

Example 3: Pediatric Study Design Objectives:
Primary purpose:
1) To estimate the recommended phase 2 dose (RP2D) of NIS793 as a single agent in children with recurrent or refractory solid tumors.
2) Define and explain the toxicity of NIS793 alone and in conjunction with chemotherapy.
3) To characterize the pharmacokinetics of NIS793 alone and in conjunction with chemotherapy in children with relapsed or refractory solid tumors.
Secondary purpose:
4) To define the preliminary antitumor activity of NIS793 alone in patients with relapsed refractory solid tumors.
5) To obtain the first phase 2 efficacy data on the antitumor activity of NIS793 in combination with cyclophosphamide and topotecan in pediatric patients with relapsed or refractory neuroblastoma.
6) To estimate the proportion of patients with recurrent measurable osteosarcoma treated with NIS793 in combination with gemcitabine who are recurrence-free at 24 weeks (EF%).
Experimental Purpose: Exploratory Purpose:
7) To explore the immunogenicity of NIS793 in this population.
8) Exploring biomarkers of NIS793 activity, including ctDNA, RNAseq tumor samples for TGFβ signatures.

Required omitted eligibility criteria:
Inclusion criteria:
age:
- Phase I cohort: Patients must be older than 12 months and 21 years or younger at the time of study entry.
- Neuroblastoma Expansion Cohort: Patients must be older than 12 months and 21 years or younger at the time of study entry.
- Osteosarcoma Expansion Cohort: Patients must be 12 months or older and 39 years or younger at the time of study entry.
Diagnosis: At initial diagnosis or recurrence, patients must have histological confirmation of malignancy.
- Phase I portion: Patients with relapsed or refractory solid tumors are eligible; patients with primary CNS tumors are excluded.
・Spread of neuroblastoma: Patients with relapsed or refractory neuroblastoma ・Spread of osteosarcoma: Patients with relapsed or refractory osteosarcoma Disease status:
- Phase 1 part: Patients must have measurable or evaluable disease.
- Neuroblastoma: Patients must have measurable disease or MIBG evaluable disease at the time of enrollment.
- Osteosarcoma cohort: Patients must have measurable disease at the time of enrollment.
- Patients with untreated disease in the brain parenchyma are not eligible.
Performance level: Karnofsky 50% or higher for patients over 16 years of age and Lansky 50 or higher for patients under 16 years of age. [Note: Patients who are unable to walk due to paralysis but are able to sit up in a wheelchair will be considered ambulatory for purposes of evaluating performance scores].
Previous treatment:
- Patients must have fully recovered from the acute toxic effects of all previous anti-cancer therapies and have completed a minimum period of time from previous anti-cancer therapy prior to enrollment.
-Patients must have no prior exposure to specific anti-TGFβ therapy.
- Patients with neuroblastoma enrolled in the expansion cohort may have previously received cyclophosphamide and topotecan.
- Patients with osteosarcoma may have previously received gemcitabine.
Required standards for organ function:
Adequate bone marrow function is defined as;
For patients with solid tumors without known bone marrow involvement:
・Peripheral blood absolute neutrophil count (ANC) ≧1000/mm3
- Platelet count calculation - 100,000/mm3 (unrelated to blood transfusion, defined as not having received a platelet transfusion for at least 7 days prior to enrollment)
Patients with known bone marrow metastatic disease are eligible for the study, provided they meet a complete blood count (and have not received a blood transfusion, provided they are not known to be refractory to red blood cell or platelet transfusions). good). These patients are not evaluable for hematological toxicity. At least 2 of every cohort of 3 patients must be evaluable for hematologic toxicity in the dose escalation portion of the study. If dose-limiting hematologic toxicity is identified on dose escalation, all subsequent patients enrolled in the dose escalation portion must be evaluable for hematologic toxicity.
Adequate renal function is defined as:
Creatinine clearance or radioisotope GFR ≧70ml/min/ ^1.73m2 or age/gender based serum creatinine is:

Adequate liver function is defined as:
- Bilirubin (total of conjugated + unconjugated types) ≦ Upper limit of reference value for age (ULN) x 1.5.
・ALT(SGPT)≦135U/L. For the purpose of this study, the UNL of ALT is 45 U/L.
-Serum albumin ≧2g/dL.
Adequate cardiac function is defined as:
- Left ventricular diameter shortening rate - Left ventricular diameter shortening rate >27% and left ventricular ejection fraction >50%.
- No clinically significant cardiac arrhythmia, stroke, or myocardial infarction within 6 months prior to enrollment.
・QTc≦480ms
- Hypertension must be well controlled with stable medication for at least 2 weeks.
Informed Consent: All patients and/or their parents or guardians must sign a written informed consent. Consent will be obtained where appropriate and in accordance with institutional guidelines.

Exclusion criteria:
Pregnant or breastfeeding:
Pregnant or lactating women cannot participate in this study as there is no information yet available regarding human fetal or teratogenic toxicity. Women who have reached menarche must take a pregnancy test. Men or women of reproductive potential have not consented to use two effective contraceptive methods, such as medically approved barrier or contraceptive methods (e.g., male or female condoms), during the study period Do not participate unless Abstinence is an acceptable method of contraception.
Concomitant Medications Corticosteroids: Patients on corticosteroids who have not received a stable or decreasing dose of corticosteroids for at least 7 days prior to enrollment are not eligible. At least 14 days must elapse since the last administration of corticosteroids when used to alleviate immunological adverse events associated with previous corticosteroids.
Clinical Trials: Patients currently receiving another investigational drug are not eligible.
Anticancer drugs: Patients currently receiving other anticancer drugs are not eligible [with the exception of leukemia patients receiving hydroxyurea, which may be continued up to 24 hours before starting protocol therapy].
Post-transplant anti-GVHD agents: Patients receiving cyclosporine, tacrolimus, or drugs to prevent graft-versus-host disease after bone marrow transplantation are not eligible for this trial.
Infections: Patients with uncontrolled infections are not eligible.
Patients who have previously received a solid organ transplant are not eligible.
Patients who, in the opinion of the investigator, may be unable to comply with the safety monitoring requirements of the trial are not eligible.
Patients with severe non-healing wounds (CTCAE grade ≧3 wound complications, dehiscence, or wound infections).
Patients with stroke or transient ischemic attack, or other ischemic event, or thromboembolic event within 3 months of starting study treatment.

Proposed clinical trial design:
Clinical trial stage:
This is a trial of single agent NIS793 in relapsed/refractory solid tumors in an expansion cohort of NIS793 with combination therapy in relapsed/refractory neuroblastoma or osteosarcoma. Phase I of the trial is a single-agent dose-finding phase using a cyclical six-part design. PK expansion is performed in up to 6 patients to obtain PK for at least 6 patients under 12 years of age and 6 patients over 12 years of age. The primary endpoint of Phase I is the incidence of dose-limiting toxicity (DLT) during the first treatment cycle of treatment. A treatment cycle is defined as 21 days (3 calendar weeks).

Standard PEP-CTN definitions for dose-limiting toxicity are used to define MTD or RP2D. Briefly, AEs and abnormal labs that result in failure to meet criteria for retreatment or inability to begin a new cycle of therapy within 7 days of the scheduled start date of the new cycle. A DLT is defined as an AE or abnormal laboratory value that is suspected to be related to therapy with NIS 793, such as a value (ie, assessed as unrelated to exacerbation, intervening disease, or concomitant medications). Although DLTs can occur during any cycle of NIS793 treatment, for purposes of RP2D dose escalation and decisions, only DLTs that occur during the first 3 weeks will be considered for decisions regarding dose titration.

The first cohort in the dose-finding phase will be treated with starting doses of 45 mg/kg (BW < 20 kg), 30 mg/kg (BW 20-40 kg) and 20 mg/kg (BW > 40 kg) every 3 weeks. This dose was determined by pharmacokinetic modeling to target exposure in adults receiving the recommended dose. If this starting dose level of NIS793 exceeds the MTD (more than 2/6 patients or more than 33% of patients with DLT in the dose confirmation and PK cohorts), then dose level -1 (30% dose reduction) ) is evaluated. If Dose Level 1 does not exceed the MTD, R2PD is declared and further dose escalation will not be sought. If Dose Level-1 exceeds the MTD, the trial will be paused to assess pharmacokinetics and investigate any toxicities to determine whether alternative doses or schedules should be explored by protocol modification. Ru.

For up to 35 cycles (approximately 2 years), unacceptable toxicity, confirmed death due to disease progression, or for any other reason (i.e., missed follow-up, subject/parent/guardian decision, or withdrawal of consent) ) Patients will continue on treatment until study treatment is discontinued due to

The recommended dose of NIS793 will then be used in an expansion cohort in conjunction with cyclophosphamide and topotecan in patients with neuroblastoma or in conjunction with gemcitabine in patients with osteosarcoma. The expansion phase consists of two cohorts: relapsed refractory osteosarcoma and relapsed/refractory neuroblastoma. Tolerability will be monitored separately in each cohort using an OBIN design.

Patients with neuroblastoma will be treated with NIS793's RP2D from the Phase 1 portion in combination with cyclophosphamide and topotecan. The primary endpoint is objective response using RECISTv1.1 in patients with measurable disease or MIBG Curie scoring for patients with MIBG evaluable disease. Objective response is complete or partial response. Simon's two-stage design is used (10+10).

Patients with osteosarcoma have NIS793 in combination with gemcitabine. We use a 9+15 Simon two-stage optimal design. The null hypothesis that the true EFS rate is 0.12 is tested against a one-sided opposition. Nine patients will be added in the first phase. If one or more of the nine evaluable patients as defined in Section 11.4 are recurrence-free after 24 weeks (8 cycles), the trial will be discontinued and the drug will be We conclude that this drug does not elicit a significant response. If at least 2 patients are recurrence-free after 24 weeks, then 15 additional patients will be added for a total of 24 patients evaluable for response. Phase 2 will not begin until at least 2 responder-evaluable patients have been relapse-free for 24 weeks. Enrollment in Phase 1 will be suspended until all patients reach the 24 week assessment or until at least 2 patients reach the 24 week assessment without an event. Relapse-free status for each patient is defined by the patient's status 24 weeks after starting therapy.

For up to 35 cycles (approximately 2 years), unacceptable toxicity, confirmed death due to disease progression, or for any other reason (i.e., missed follow-up, subject/parent/guardian decision, or withdrawal of consent) ) Patients will continue on treatment until discontinuation of study treatment.

Clinical trial scheme

Estimated Sample Size (Explanation of Estimates): Study up to 4 patients and up to 18 patients (6 dose level 1, 6 dose level -1, 6 PK expansion) to determine RP2D and pharmacokinetics of NIS793. The single agent dosing portion of the procedure is performed. Single dose dose tapering is incorporated. An additional 10-20 patients with neuroblastoma may be enrolled to determine the activity of NIS793 in conjunction with cyclophosphamide and topotecan. A minimum of 9 and up to 24 patients with osteosarcoma may receive NIS793 in conjunction with gemcitabine (up to 62 patients).

Treatment plan:
During Phase I dose titration, NIS793 administration will be initiated at dose level 1 and tapered if necessary.
- NIS793 45 mg/kg (BW < 20 kg), 30 mg/kg (BW 20-40 kg) and 20 mg/kg (BW > 40 kg) every 3 weeks (additional/intermediate dose levels or less frequent dosing may be explored) .
Once the RP2D of NIS793 is determined, an expansion cohort will begin.
Patients in the neuroblastoma cohort:
・NIS793 in RP2D
- Cyclophosphamide: 250 mg/m ² /dose intravenously x 5 days, days 1 to 5 - Topotecan: 0.75 mg/m ² /dose intravenously x 5 days, administered on days 1 to 5.
Patients with osteosarcoma have
・NIS793 in RP2D
- Gemcitabine: 675 mg/m ² /dose administered on days 1 and 8.
One treatment cycle is defined as 21 days.
Disease evaluation is performed every 2 cycles 5 times, then every 4 cycles. The maximum number of cycles is 35 (approximately 2 years).

Equivalents While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art from consideration of this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims (101)

A method of treating a proliferative disease in a subject in need thereof, the method comprising administering to said subject a TGFβ antibody and at least one additional therapeutic agent, wherein said TGFβ antibody A method, wherein the dose is about 1400 to about 2100 mg or about 20 to about 45 mg/kg, administered once every three weeks. 2. The method of claim 1, wherein the TGFβ antibody is administered once every two weeks at about 1400 mg. 2. The method of claim 1, wherein the TGFβ antibody is administered once every two weeks at about 30 mg/kg. 2. The method of claim 1, wherein the TGFβ antibody is administered at about 2100 mg once every 2, 3 or 4 weeks. 2. The method of claim 1, wherein the TGFβ antibody is administered once every three weeks at about 20 mg/kg. 2. The method of claim 1, wherein the TGFβ antibody is administered once every three weeks at about 45 mg/kg. From claim 1, wherein the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. 6. The method according to any one of 6. 8. The method of any one of claims 1 to 7, wherein the TGFβ antibody comprises a heavy chain variable region and a light chain variable region shown in amino acid sequence number 7 and 8, respectively. 9. The method according to any one of claims 1 to 8, wherein the TGFβ antibody comprises a heavy chain variable region and a light chain variable region shown in the amino acid sequences of SEQ ID NOs: 9 and 10, respectively. 10. The method according to any one of claims 1 to 9, wherein the TGFβ antibody is a monoclonal antibody. 11. The method of any one of claims 1 to 10, wherein the TGFβ antibody is a fully human antibody. 12. The method of any one of claims 1-11, wherein the TGFβ antibody is administered over a period of about 30 minutes. The additional therapeutic agents include PD-1 inhibitors, gemcitabine, nab-paclitaxel, folinic acid (leucovorin calcium), levoleucovorin, fluorouracil (5-FU), oxaliplatin (eloxatin), bevacizumab, irinotecan, cyclophosphamide. 13. The method according to any one of claims 1 to 12, wherein the drug is one or more of , capecitabine, or topotecan. 14. The method of any one of claims 1-13, wherein the additional therapeutic agent comprises a PD-1 inhibitor. 15. The method of claim 14, wherein the PD-1 inhibitor is an anti-PD-1 antibody. 16. The method of claim 15, wherein the anti-PD-1 antibody is PDR001 (spartalizumab), BGB-A317 (tislelizumab), or BGB-108. 17. The method of claim 16, wherein the anti-PD-1 antibody is tislelizumab. 18. The method of any one of claims 15 to 17, wherein the anti-PD-1 antibody is administered at 300 mg per 28 day cycle or 200 mg per 21 day cycle. 19. The method of any one of claims 1-18, wherein the additional therapeutic agent comprises gemcitabine. 20. The method of claim 19, wherein gemcitabine is administered at about 1000 mg/ ^m2 . 21. The method of claim 20, wherein gemcitabine is administered on days 1, 8, and 15 of a 28 day cycle. 20. The method of claim 19, wherein gemcitabine is administered at 675 mg/ ^m2 . 23. The method of any one of claims 19 to 22, wherein gemcitabine is administered on days 1 and 8 of a 21 day cycle. 24. The method of any one of claims 1-23, wherein the additional therapeutic agent comprises nab-paclitaxel. 25. The method of claim 24, wherein nab-paclitaxel is administered at about 125 mg/ ^m2 . 26. The method of claim 24 or 25, wherein nab-paclitaxel is administered on days 1, 8, and 15 of a 28 day cycle. 27. The method of any one of claims 1-26, wherein the additional therapeutic agent comprises gemcitabine and nab-paclitaxel. 28. The method of any one of claims 1-27, wherein the additional therapeutic agent comprises a PD-1 inhibitor, gemcitabine and nab-paclitaxel. 29. The method of any one of claims 1-28, wherein the additional therapeutic agent comprises bevacizumab. 30. The method of claim 29, wherein bevacizumab is administered at about 5 mg/kg. 31. The method of claim 29 or 30, wherein bevacizumab is administered on days 1 and 15 of a 28 day cycle. 32. The method of any one of claims 1-31, wherein the additional therapeutic agent comprises 5-fluorouracil. 33. The method of claim 32, wherein 5-fluorouracil is administered at about 400 mg/ ^m2 . 33. The method of claim 32, wherein 5-fluorouracil is administered at about 2400 mg/ ^m2 . 35. The method of any one of claims 32 to 34, wherein 5-fluorouracil is administered on days 1 and 15 of a 28 day cycle. 36. The method of any one of claims 32 to 35, wherein 5-fluorouracil is administered as an intravenous bolus. 5-Fluorouracil is administered as an intravenous bolus at 400 mg/ ^m2 on days 1 and 15 of each 28-day cycle, followed by a continuous intravenous infusion at 2400 mg/ ^m2 for 46 hours. 37. A method according to any one of claims 32 to 36. 38. The method of any one of claims 1-37, wherein the additional therapeutic agent comprises leucovorin or levoleucovorin. 39. The method of claim 38, wherein leucovorin or levoleucovorin is administered at about 400 mg/ ^m2 or 200 mg/ ^m2 , respectively. 40. The method of claim 38 or 39, wherein leucovorin or levoleucovorin is administered on days 1 and 15 of a 28 day cycle. 41. The method of any one of claims 1-40, wherein the additional therapeutic agent comprises oxaliplatin. 42. The method of claim 41, wherein oxaliplatin is administered at about 85 mg/ ^m2 . 43. The method of claim 41 or 42, wherein oxaliplatin is administered on days 1 and 15 of a 28 day cycle. 44. The method of any one of claims 1-43, wherein the additional therapeutic agent comprises irinotecan. 45. The method of claim 44, wherein irinotecan is administered at about 180 mg/ ^m2 . 46. The method of claim 44 or 45, wherein irinotecan is administered on days 1 and 15 of a 28 day cycle. 47. The method of any one of claims 1-46, wherein the additional therapeutic agent comprises bevacizumab, 5-fluorouracil, leucovorin (or levoleucovorin), and oxaliplatin. 48. The method of any one of claims 1-47, wherein the additional therapeutic agent comprises a PD-1 inhibitor, bevacizumab, 5-fluorouracil, leucovorin (or levoleucovorin), and oxaliplatin. 47. The method of any one of claims 1-46, wherein the additional therapeutic agent comprises bevacizumab, 5-fluorouracil, leucovorin, and irinotecan. 50. The method of any one of claims 1-46 and 49, wherein the additional therapeutic agent comprises a PD-1 inhibitor, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan. 51. The method of any one of claims 1-50, wherein the TGFβ antibody and the additional therapeutic agent are administered to the subject in two or more cycles. 52. The method of any one of claims 1-51, wherein the proliferative disease is pancreatic cancer or colorectal cancer. 53. The method of any one of claims 1-52, wherein the additional therapeutic agent comprises cyclophosphamide and topotecan. 54. The method of claim 53, wherein cyclophosphamide is administered at 250 mg/ ^m2 . 55. The method of claim 53 or 54, wherein cyclophosphamide is administered for 5 days. 56. The method of claim 55, wherein the five days are consecutive days. 57. The method of any one of claims 53 to 56, wherein topotecan is administered at 0.75 mg/ ^m2 . 58. The method of any one of claims 53-57, wherein topotecan is administered for 5 days. 59. The method of claim 58, wherein the five days are consecutive days. 60. The method of any one of claims 1-59, wherein the proliferative disease is neuroblastoma or osteosarcoma. 61. The method of any one of claims 1-60, wherein the subject is a pediatric patient. A method of treating pancreatic adenocarcinoma in a subject in need thereof, the method comprising administering to said subject 2100 mg of TGFβ antibody, 1000 mg/m ² of gemcitabine, and 125 mg/m ² of nab-paclitaxel, wherein said TGFβ antibody , on days 1, 8, and 15 of a 28-day cycle, and the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and SEQ ID NO: 4, 5, and 6 each of light chain CDR1, CDR2, and CDR3. 63. The method of claim 62, further comprising administering to the patient a PD-1 inhibitor. 64. The method of claim 63, wherein the PD-1 inhibitor is spartalizumab or tislelizumab. 65. The method of claim 64, wherein the PD-1 inhibitor is spartalizumab and is administered at 400 mg once every four weeks. 65. The method of claim 64, wherein the PD-1 inhibitor is tislelizumab and is administered weekly at 100 mg. 67. The method of claim 66, wherein the tislelizumab is administered at 400 mg once every four weeks. 68. The method of any one of claims 62-67, wherein the TGFβ antibody, gemcitabine and nab-paclitaxel (and optionally a PD-1 inhibitor) are administered to the subject in two or more cycles. 69. The method of any one of claims 62-68, wherein the TGFβ antibody, gemcitabine and nab-paclitaxel (and optionally a PD-1 inhibitor) are administered intravenously to the subject. A method of treating colorectal cancer in a subject in need thereof, the method comprising: TGFβ antibody 2100 mg, bevacizumab 5 mg/kg, 5-fluorouracil 400-2400 mg/m ² , levoleucovorin 400 mg/m ² , and oxaliplatin 85 mg/m 2 ² to the subject, wherein the TGFβ antibody, bevacizumab, 5-fluorouracil, levoleucovorin, and oxaliplatin are administered on days 1, 8, and 15 of a 28-day cycle, and the TGFβ antibody, A method comprising heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively. 71. The method of claim 70, wherein the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin are administered to the subject in two or more cycles. 72. The method of claim 70 or 71, wherein the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and oxaliplatin are administered intravenously to the subject. 73. The method of any one of claims 70 to 72, wherein 5-fluorouracil is administered as an intravenous bolus at 400 mg/ ^m2 , followed by a continuous intravenous infusion for 46 hours at 2400 mg/ ^m2 . . A method of treating colorectal cancer in a subject in need thereof, the method comprising: 2100 mg of TGFβ antibody, 5 mg/kg of bevacizumab, 400-2400 mg/m ² of 5-fluorouracil, 400 mg/m ² of leucovorin, and 180 mg/m ² of irinotecan. administering to the subject, the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan are administered on days 1 and 15 of a 28-day cycle, and the TGFβ antibody comprises SEQ ID NO: 1; 2 and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively. 75. The method of claim 74, wherein the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan are administered to the subject in two or more cycles. 76. The method of claim 74 or 75, wherein the TGFβ antibody, bevacizumab, 5-fluorouracil, leucovorin, and irinotecan are administered intravenously to the subject. 77. The method of any one of claims 74 to 76, wherein 5-fluorouracil is administered as an intravenous bolus at 400 mg/ ^m2 , followed by a continuous intravenous infusion for 46 hours at 2400 mg/ ^m2 . . A method of treating neuroblastoma in a subject in need thereof, the method comprising: administering 20, 30, or 45 mg/kg of TGFβ antibody, 250 mg/m ² of cyclophosphamide, and 0.75 mg/m ² of topotecan to said subject. wherein said TGFβ antibody is administered every three weeks, and cyclophosphamide and topotecan are administered on days 1 to 5 of a 21 day cycle, and said TGFβ antibody is administered with SEQ ID NO: 1 , 2, and 3, and the light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively. A method of treating osteosarcoma in a subject in need thereof, comprising administering to said subject 20, 30, or 45 mg/kg of TGFβ antibody and 675 mg/m ² of gemcitabine, wherein said TGFβ antibody is weekly, and gemcitabine is administered on days 1 and 8 of a 21-day cycle, and the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 1, 2, and 3, respectively; and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 4, 5, and 6, respectively. 80. The method of claim 78 or 79, wherein the subject has a body weight of less than 20 kg and 45 mg/kg of the TGFβ antibody is administered. 80. The method of claim 78 or 79, wherein the subject has a body weight of 20-40 kg and 30 mg/kg of the TGFβ antibody is administered. 80. The method of claim 78 or 79, wherein the subject has a weight greater than 40 kg and 20 mg/kg of the TGFβ antibody is administered. 80. The method of claim 78 or 79, wherein the subject is less than 18 years of age. 84. The method of any one of claims 1-83, wherein the TGFβ inhibitor is administered simultaneously with the additional therapeutic agent. 84. The method of any one of claims 1-83, wherein the TGFβ inhibitor is administered before the additional therapeutic agent. 84. The method of any one of claims 1-83, wherein the TGFβ inhibitor is administered after the additional therapeutic agent. 84. The method of any one of claims 1-83, wherein the TGFβ inhibitor and/or the additional therapeutic agent are administered until remission. A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, comprising:
(a) 2100 mg of TGFβ antibody,
(b) 5-fluorouracil 500 mg/m ² as an intravenous bolus followed by 2400 mg/m ² by continuous infusion for 46 hours;
(c) 400 mg/m ² of icovorin (folinic acid) intravenously or 200 mg/m ² of levoleucovorin intravenously;
(d) irinotecan 80 mg/m ² intravenously; and (e) bevacizumab 5 mg/kg.
comprising administering to said subject,
The TGFβ antibody is administered on day 1 (and optionally day 15) of a 28-day cycle, and 5-fluorouracil, leucovorin (or levoleucovorin), oxaliplatin, and bevacizumab are administered on days 1 and 15 of a 28-day cycle. day, and the TGFβ antibody has heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. including methods. A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising:
(a) 2100 mg of TGFβ antibody,
(b) 5-fluorouracil 500 mg/m ² as an intravenous bolus followed by 2400 mg/m ² by continuous infusion for 46 hours;
(c) 400 mg/m ² of leucovorin (folinic acid) intravenously or 200 mg/m ² of levoleucovorin intravenously;
(d) oxaliplatin 85 mg/m ² intravenously; and (e) bevacizumab 5 mg/kg.
comprising administering to said subject,
The TGFβ antibody is administered on day 1 (and optionally day 15) of a 28-day cycle, and 5-fluorouracil, leucovorin (or levoleucovorin), oxaliplatin, and bevacizumab are administered on days 1 and 15 of a 28-day cycle. and wherein the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 1, 2, and 3, respectively. 90. The method of claim 88 or 89, further comprising administering the PD-1 inhibitor to the patient. 91. The method of claim 90, wherein the PD-1 inhibitor is tislelizumab having a heavy chain of SEQ ID NO: 321 and a light chain of SEQ ID NO: 322. 92. The method of claim 91, wherein tislelizumab is administered intravenously at 300 mg on day 1 of each 28 day cycle. 93. Any of claims 88-92, wherein the TGFβ antibody, 5-fluorouracil, leucovorin (or levoleucovorin), oxaliplatin, irinotecan, bevacizumab, or PD-1 inhibitor is administered to the subject in two or more cycles. The method described in paragraph (1). A method of treating gastric cancer in a subject in need thereof, comprising:
(a) 2100 mg of TGFβ antibody,
(b) 130 mg/m ² of oxaliplatin intravenously;
(c) capecitabine 1000 mg/m ² orally twice a day;
comprising administering to said subject,
the TGFβ antibody is administered once every three weeks, and oxaliplatin is administered on day 1 of the Q3W cycle, and capecitabine is administered on days 1 to 14 of the Q3W cycle;
The method, wherein the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. A method of treating gastric cancer in a subject in need thereof, comprising:
(a) 2100 mg of TGFβ antibody,
(b) 130 mg/m ² of oxaliplatin intravenously;
(c) capecitabine 1000 mg/m ² orally twice a day;
comprising administering to said subject,
the TGFβ antibody is administered once every two weeks, and oxaliplatin is administered on day 1 of the Q3W cycle, and capecitabine is administered on days 1 to 14 of the Q3W cycle;
The method, wherein the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. A method of treating gastric cancer in a subject in need thereof, comprising:
(a) 2100 mg of TGFβ antibody,
(b) 85 mg/m ² of oxaliplatin intravenously;
(c) Leucovorin (folinic acid) 400 mg/m ² intravenously or Levoleucovorin 200 mg/m ² intravenously, and (d) 5-fluorouracil 400 mg/m ² intravenously bolus per day. , followed by administering to said subject 1200 mg/ ^m2 by intravenous administration,
The TGFβ antibody is administered once every three weeks, and oxaliplatin, leucovorin (or levoleucovorin), 5-fluorouracil is administered on day 1 of the Q2W cycle, and the 1200 mg is administered on days 1-2 of the Q2W cycle. Administered intravenously into the eye,
The method, wherein the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. A method of treating gastric cancer in a subject in need thereof, comprising:
(a) 2100 mg of TGFβ antibody,
(b) 85 mg/m ² of oxaliplatin intravenously;
(c) 400 mg/m ² of leucovorin (folinic acid) intravenously or 200 mg/m ² of levoleucovorin intravenously;
(d) 5-fluorouracil as an intravenous bolus of 400 mg/m ² followed by 1200 mg/m ² per day.
comprising administering to said subject,
The TGFβ antibody is administered once every two weeks, and oxaliplatin, leucovorin (or levoleucovorin), 5-fluorouracil is administered on day 1 of the Q2W cycle, and the 1200 mg is administered on days 1-2 of the Q2W cycle. Administered intravenously into the eye,
The method, wherein the TGFβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 4, 5, and 6, respectively. 98. The method of claim 94 or 97, further comprising administering to said patient a PD-1 inhibitor. 99. The method of claim 98, wherein the PD-1 inhibitor is tislelizumab having a heavy chain of SEQ ID NO: 321 and a light chain of SEQ ID NO: 322. 100. The method of claim 99, wherein tislelizumab is administered intravenously at 300 mg on day 1 of each 28 day cycle. 100. The method of claim 99, wherein tislelizumab is administered intravenously at 200 mg once every three weeks.