CN117580589A - Combination of anti-galectin-9 antibodies and chemotherapeutic agents for use in cancer therapy - Google Patents

Combination of anti-galectin-9 antibodies and chemotherapeutic agents for use in cancer therapy Download PDF

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CN117580589A
CN117580589A CN202280045695.8A CN202280045695A CN117580589A CN 117580589 A CN117580589 A CN 117580589A CN 202280045695 A CN202280045695 A CN 202280045695A CN 117580589 A CN117580589 A CN 117580589A
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antibody
subject
dose
galectin
administered
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A·菲利波维奇
E·艾伦科
H·帕登
C·科思
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Pure Technology Lyt Co ltd
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Pure Technology Lyt Co ltd
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Priority claimed from PCT/US2022/027142 external-priority patent/WO2022232653A1/en
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Abstract

Disclosed herein are combination therapies for treating solid tumors (e.g., pancreatic Ductal Adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal Cell Carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other gastrointestinal solid tumors) comprising an antibody that binds human galectin-9 (anti-Gal 9 antibody, e.g., G9.2-17) and one or more chemotherapeutic agents, e.g., gemcitabine, paclitaxel, or a combination thereof.

Description

Combination of anti-galectin-9 antibodies and chemotherapeutic agents for use in cancer therapy
Cross Reference to Related Applications
The present application claims the benefit of U.S. c. ≡119 (e) from U.S. provisional application nos. 63/182,519, 2021, 5, 26, and 2022, 25, each of which is incorporated herein by reference in its entirety.
Sequence listing
The present application contains a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 25 of 4 months 2022, named 112174-0212-NP010WO1_SEQ. Txt, and was 89,162 bytes in size.
Background
Galectin-9 is a tandem repeat lectin consisting of two Carbohydrate Recognition Domains (CRDs) and was first discovered and described in 1997 in patients with Hodgkin Lymphoma (HL) (Tureci et al, J.biol. Chem.1997,272, 6416-6422). Three isoforms exist and may be located either intracellular or extracellular. Elevated levels of galectin-9 have been observed in a variety of cancers, including melanoma, hodgkin lymphoma, hepatocellular carcinoma, pancreatic cancer, gastric cancer, colon cancer, and clear cell renal cell carcinoma (Wdowiak et al, int.j.mol. Sci.2018,19,210). In renal cancer, patients with high galectin-9 expression showed more advanced disease progression with larger tumor sizes (Kawashima et al; BJU int.2014; 113:320-332). In Melanoma, galectin-9 was expressed in 57% of the tumors and significantly increased in the plasma of patients with advanced Melanoma compared to healthy controls (Enning ga et al, melanoma Res.2016, 10 months; 26 (5): 429-441). Many studies have shown the utility of galectin-9 as a prognostic marker and have recently also shown its utility as a potential new drug target (Enning et al 2016; kawashima et al BJU Int 2014;113:320-332; kageshita et al, int J cancer.2002, 20 th day; 99 (6): 809-16, and references therein).
Galectin-9 has been described to play an important role in many cellular processes such as adhesion, cancer cell aggregation, apoptosis and chemotaxis. Recent studies have shown that galectin-9 plays a role in supporting immunomodulation of tumors, e.g., by down-regulating Th 1-type responses, th2 polarization, and polarization of macrophages to the M2 phenotype. This work also included studies that showed that galectin-9 was involved in the direct inactivation of T cells through interaction with T cell immunoglobulin and mucin 3 (TIM-3) receptors (Dardalkon et al, J Immunol.,2010,185,1383-1392; sanchez-Fuleyo et al, nat Immunol.,2003,4,1093-1101).
Galectin-9 has also been found to play a role in polarizing T cell differentiation towards tumor suppression phenotypes and promoting tolerogenic macrophage programming and adaptive immunosuppression (Daley et al, nat med.,2017,23,556-567). In a mouse model of Pancreatic Ductal Adenocarcinoma (PDAC), blocking checkpoint interactions between galectin-9 found on innate immune cells in the Tumor Microenvironment (TME) has been shown to increase anti-tumor immune responses in TME and slow tumor progression (Daley et al, nat med.,2017,23,556-567). Galectin-9 was also found to bind to CD206 (a surface marker of M2 type macrophages) resulting in decreased secretion of CVL22 (MDC), a macrophage derived chemokine associated with longer survival and lower risk of recurrence of lung cancer (Enning ga et al J Pathol.2018, month 8; 245 (4): 468-477).
Disclosure of Invention
The present disclosure is based on the unexpected discovery that synergy was observed in a combination therapy involving an exemplary anti-galectin 9 antibody (e.g., G9.2-17 (IgG 4)) and a chemotherapeutic agent such as gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel or nab-paclitaxel) in an animal model. Alternatively or additionally, the present disclosure is based at least in part on the unexpected discovery that: the anti-galectin-9 antibody G9.2-17 (IgG 4) has a faster clearance rate in human subjects compared to other antibody therapeutics. Thus, a treatment regimen comprising a weekly dosing schedule was developed to ensure a suitable plasma concentration of anti-galectin 9 antibody (anti-Gal-9 antibody) for achieving a therapeutic effect, such as therapeutic systemic exposure levels.
Accordingly, provided herein are methods for treating solid tumors involving an anti-galectin-9 antibody (e.g., G9.2-17 or a functional variant thereof) and one or more chemotherapeutic agents (e.g., gemcitabine, paclitaxel such as protein-bound paclitaxel (e.g., nab-paclitaxel or a functional variant thereof)) Or a combination thereof). In some cases, an anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG 4), can be administered on a weekly dosing regimen.
In some embodiments, the methods disclosed herein for treating solid tumors can comprise administering to a subject in need thereof an effective amount of an antibody that binds human galectin-9 (anti-Gal-9 antibody). The anti-Gal-9 antibody can have the same heavy chain Complementarity Determining Regions (CDRs) and the same light chain CDRs as antibody G9.2-17. The subject may receive an anti-cancer therapy comprising one or more chemotherapeutic agents.
In some embodiments, the methods disclosed herein for treating solid tumors can comprise administering to a subject in need thereof an effective amount of an antibody that binds human galectin-9 (anti-Gal-9 antibody) and an effective amount of one or more chemotherapeutic agents. The anti-Gal-9 antibody can have the same heavy chain Complementarity Determining Regions (CDRs) and the same light chain CDRs as antibody G9.2-17.
In some embodiments, the methods disclosed herein for treating a solid tumor may comprise administering to a subject in need thereof an effective amount of one or more chemotherapeutic agents. The subject may receive a therapy comprising an antibody that binds human galectin-9 (anti-Gal-9 antibody) having the same heavy chain Complementarity Determining Regions (CDRs) and the same light chain CDRs as antibody G9.2-17.
Any of the methods disclosed herein can be used to treat metastatic solid tumors. In some examples, the solid tumor is Pancreatic Ductal Adenocarcinoma (PDAC), such as metastatic PDAC.
In some embodiments, a subject to be treated by any of the methods disclosed herein can have one or more of the following features: (i) no resectable cancer; (ii)
Is not infected by SARS-CoV-2; and (iii) no active brain or leptomeningeal metastasis. In some examples, the solid tumor is Pancreatic Ductal Adenocarcinoma (PDAC), and the subject is free of locally advanced PDACs that are free of distant organ metastatic deposits.
In some embodiments, the one or more chemotherapeutic agents to which any of the methods disclosed herein relate may comprise an antimetabolite (e.g., a nucleoside analog), a microtubule inhibitor, or a combination thereof. In some examples, the nucleoside analog is gemcitabine and/or the tubulin inhibitor is paclitaxel, e.g., nanoparticulate albumin-bound paclitaxel (e.g.,)。
in some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2mg/kg to about 32mg/kg (e.g., about 0.2mg/kg to about 16mg/kg, 0.5mg/kg to about 16mg/kg, about 2mg/kg to about 32mg/kg, or about 2mg/kg to about 16mg/kg, or about 0.2mg/kg to about 15mg/kg, or about 0.2mg/kg to about 16mg/kg or higher).
In some embodiments, the anti-Gal-9 antibody is administered to the subject weekly. In some embodiments, the anti-Gal-9 antibody may be administered to the subject at a dose of about 0.2mg/kg to about 32mg/kg once a week. In some embodiments, the anti-Gal-9 antibody may be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. Alternatively, an anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG 4), can be administered to a subject at a dose of about 650mg to about 1120mg once a week. For example, the anti-Gal-9 antibody may be administered to a subject at a dose of 10mg/kg once a week or at a plateau dose of about 650-700mg once a week. Alternatively, the anti-galectin-9 antibody may be administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of about 1040-1120mg once a week.
In some embodiments, the anti-Gal-9 antibody is administered to the subject every 2 or 3 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 0.2mg/kg, 0.6mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, or 16mg/kg or higher. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from 2mg/kg, 4mg/kg, 8mg/kg, 12mg/kg, or 16mg/kg or higher. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from the group consisting of 0.2mg/kg, 0.6mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg or higher. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from 0.2mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, or 16mg/kg once every 2 weeks. In some embodiments, the antibody is administered once every 2 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from 2mg/kg, 4mg/kg, 8mg/kg, 12mg/kg, or 16mg/kg or higher once every 2 weeks. In some embodiments, the antibody is administered once every 2 weeks. In some embodiments, the anti-galectin-9 antibody is administered to the subject at a dose selected from 0.2mg/kg, 0.6mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg or more once every 2 weeks.
In some embodiments, the anti-Gal-9 antibody, such as G9.2-17 (IgG 4), may be administered to the subject at a dose of about 650mg to about 1120mg once every 2-6 weeks (e.g., once every 2 weeks, once every 3 weeks, or once every 4 weeks). In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 650mg to about 700mg once every 2-6 weeks (e.g., once every 2 weeks, once every 3 weeks, or once every 4 weeks). In other examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 1040mg to about 1120mg every 2-6 weeks (e.g., every 2 weeks, every 3 weeks, or every 4 weeks).
In some embodiments, the anti-Gal-9 antibody is administered once every 2 weeks for one period, once every 2 weeks for two periods, once every 2 weeks for 3 periods, once every 2 weeks for 4 periods, or once every 2 weeks for more than 4 periods. In some embodiments, the duration of treatment is 0-3 months, 0-6 months, 3-6 months, 6-12 months, 12-24 months, or longer. In some embodiments, the duration of treatment is 12-24 months or longer. In some embodiments, the period extends for a duration of 3 months to 6 months or 6 months to 12 months or 12 months to 24 months or more. In some embodiments, the cycle length is modified, e.g., temporarily or permanently, to a longer duration, e.g., 3 weeks or 4 weeks or 6 weeks.
In some embodiments, the anti-Gal-9 antibody is administered to the subject by intravenous infusion. In some embodiments, the cancer is PDAC. In some embodiments, the cancer is a metastatic cancer. In some examples, multiple doses of anti-galectin 9 antibody may be administered to a subject, with later doses being higher than earlier doses.
In some embodiments, the anti-Gal-9 antibody may be administered to the subject by intravenous infusion at a dose of about 0.5mg/kg to about 32mg/kg once every two weeks. In some embodiments, the anti-Gal-9 antibody may be administered to the subject by intravenous infusion at a dose of about 0.2mg/kg to about 32mg/kg once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 0.5mg/kg by intravenous infusion once every two weeks. In some embodiments, the anti-Gal-9 antibody may be administered to the subject by intravenous infusion at a dose of about 2mg/kg to about 16mg/kg once every two weeks. In some embodiments, the anti-Gal-9 antibody may be administered to the subject at a dose of about 0.2mg/kg to about 16mg/kg or more once every two weeks by intravenous injection. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 0.2mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 0.6mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 0.63mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 2mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 4mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 6mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 6.3mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 8mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 10mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 12mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 16mg/kg or higher by intravenous infusion once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at a dose of about 32mg/kg by intravenous infusion once every two weeks. In some embodiments, the anti-Gal-9 antibody may be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week.
In any of the methods disclosed herein, the one or more chemotherapeutic agents comprise an antimetabolite, a microtubule inhibitor, or a combination thereof. For example, the antimetabolite may be gemcitabine. Alternatively or additionally, the microtubule inhibitor may be paclitaxel. In some cases, the paclitaxel is protein-bound paclitaxel, e.g., nanoparticle albumin-bound paclitaxel. In some embodiments, the one or more chemotherapeutic agents comprise a combination of gemcitabine and paclitaxel.
In some embodiments, the method comprises a 28 day cycle, wherein the anti-Gal 9 antibody is administered to the subject on days 1 and 15, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, at 125mg/m 2 Paclitaxel is administered intravenously to the subject. In some examples, at 1000mg/m 2 Gemcitabine is administered to a subject.
In some cases, the methods disclosed herein can comprise a 28 day cycle, wherein the anti-Gal-9 antibody is administered to the subject on days 1, 8, 15, and 22, and the gemcitabine and paclitaxel are administered to the subject on days 1, 8, and 15. In some examples, at 125mg/m 2 Paclitaxel is administered intravenously to the subject. Alternatively or additionally, at 1000mg/m 2 Gemcitabine is administered to a subject.
In some embodiments, an anti-galectin-9 antibody comprises a light chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 1, a light chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 2, and a light chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 3, and/or comprises a heavy chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 4, a heavy chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 5, and a heavy chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 6.
In some embodiments, an anti-Gal 9 antibody can comprise a heavy chain variable region (V H ) The method comprises the steps of carrying out a first treatment on the surface of the And a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO. 8 L ). In some examples, the anti-Gal 9 antibody may be an IgG molecule, e.g., an IgG4 molecule. In a specific example, an anti-Gal 9 antibody can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO. 19 and a light chain comprising the amino acid sequence of SEQ ID NO. 15.
In some embodiments, one or more chemotherapeutic agents may be administered to the subject on the day the subject receives an anti-galectin 9 antibody. Alternatively, the administration of one or more chemotherapeutic agents and the administration of the anti-galectin 9 antibody may be performed for two consecutive days. In some examples, administration of one or more chemotherapeutic agents may be performed prior to administration of the anti-Gal-9 antibody, e.g., on the first dosing day, and on a subsequent day, the anti-galectin 9 antibody.
In any of the methods disclosed herein, the subject may be a human patient. In some embodiments, the subject may comprise galectin-9 positive cancer cells or immune cells. In some cases, the subject may have elevated galectin-9 levels relative to a control value. For example, the subject may have elevated serum or plasma levels of galectin-9 relative to a control value.
In some embodiments, the subject may have received at least one line of systemic anticancer therapy. In other embodiments, the subject may not have received prior therapy involving gemcitabine and/or paclitaxel or has received prior therapy involving gemcitabine and/or paclitaxel at least six months prior to administration of the anti-Gal-9 antibody.
In some cases, the subject is examined for one or more of the following characteristics before, during, and/or after treatment: (a) One or more tumor markers in a tumor biopsy sample from a subject, optionally wherein the one or more tumor markers comprise CA15-3, CA-125, CEA, CA19-9, and/or alpha-fetoprotein, and any other tumor type specific tumor markers; (b) cytokine profile; and (c) galectin 9 serum/plasma levels, d) peripheral blood mononuclear cell immunophenotyping, e) tumor tissue biopsy/resection specimen multiplex immunophenotyping, f) tumor tissue biopsy/resection specimen galectin-9 expression levels and patterns, g) any other immunophenotyping test such as: PDL-1 immunohistochemistry, tumor Mutational Burden (TMB), tumor microsatellite instability status, and groups such as: in NanoString Gene expression system, 18-gene marker and PanCancer IO 360 TM Developed on measurement (NanoString Technologies) and the likeHalioDx, immunoSeq-Adaptive Biotechnologies, TIS. Other suitable biomarkers specific for the target tumor may also be examined.
Any of the methods disclosed herein can further comprise monitoring the subject for the occurrence of one or more adverse effects. In some cases, the one or more adverse effects include liver injury, blood toxicity, neurotoxicity, skin toxicity, gastrointestinal toxicity, or a combination thereof.
In some embodiments, the method may further comprise reducing the dose of the anti-Gal 9 antibody, the dose of one or more chemotherapeutic agents, or both when adverse effects are observed. For example, when moderate to severe liver injury is observed in the subject, the method can further comprise reducing the dose of anti-Gal-9 antibody, the dose of gemcitabine, the dose of paclitaxel, or a combination thereof. In one specific example, a reduction in the dosage of anti-Gal-9 antibody, or at least a 30% reduction, is performed according to the clinician's assessment. In another example, a 30% or 50% reduction level of the previous dose level is implemented. If desired, a further 30% of the dose reduction dose level-1 (the level at which the first dose is reduced) is administered (dose level-2, the level at which the second dose is reduced). In another example, another dose reduction of 50% of dose level-1 (dose level-2) is performed. In some embodiments, administration of one or more doses reduces the previous dose level by about 10% to about 80%. In some embodiments, one or more dose reductions are performed by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, or about 70% to about 80% of the previous level. In some embodiments, one or more dose reductions are performed by 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, or 70% to 80% of the previous level. In some embodiments, one or more doses are administered to reduce the previous level by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, one or more doses are administered to reduce the previous level by 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%. In some cases, administration of paclitaxel is stopped when the subject's aspartate Aminotransferase (AST) level is greater than 10 x upper normal limit (ULN), bilirubin level is greater than 5 x ULN, or both.
In some cases, the method may further comprise reducing the amount of liver damage observed when moderate to severe liver damage is observedThe dose of the anti-Gal-9 antibody, the dose of the gemcitabine, the dose of the paclitaxel, or a combination thereof. In other cases, the method may further comprise reducing the dose or terminating administration of the anti-Gal-9 antibody, the gemcitabine, the paclitaxel, or a combination thereof when severe hematologic toxicity, neurotoxicity, dermatologic toxicity, and/or gastrointestinal toxicity is observed. In some examples, the dose of paclitaxel is reduced to 100mg/m 2 -75mg/m 2 . In other examples, the dose of gemcitabine is reduced to 800mg/m 2 -600mg/m 2 。
The use of any of the pharmaceutical compositions and anti-galectin-9 antibodies used in the treatment of solid tumors (e.g., those described herein and including metastatic solid tumors) in combination with one or more chemotherapeutic agents as also disclosed herein for the preparation of a medicament for the treatment of solid tumors is also within the scope of the present disclosure.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will become apparent from the following drawings and detailed description of several embodiments, and the appended claims.
Drawings
The following drawings form a part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which aspects may be better understood by reference to the drawings in conjunction with the detailed description of the specific embodiments presented herein.
Fig. 1A-1D include graphs showing Kaplan-Meier survival curves and log rank test of orthotopic mPA6115 pancreatic cancer xenograft mouse models grouped according to treatment regimen. Group 1 = untreated; group 2 = chemical vehicle control, saline; group 3 = isotype IgG1 mice; group 4 = anti-Gal 9 mAb; group 5 = gemcitabine/Abraxane; and group 6 = anti-Gal 9 mAb and gemcitabine/Abraxane. Fig. 1A shows survival curves for all six groups. Fig. 1B shows survival curves for groups 1, 5 and 6. Fig. 1C shows survival curves for groups 1, 4 and 6. Fig. 1D shows survival curves for groups 1, 4, 5 and 6.
Fig. 2 includes graphs showing the risk ratio (HR) and its 95% confidence interval (% 95 CI) for groups 4-6 relative to groups 1, 2 and 3, respectively, calculated according to cox-regression analysis, where group 1 = untreated in situ mPA6115 mice; group 2 = chemical vehicle control, saline treated in situ mPA6115 mice; group 3 = isotype IgG1 mice treated in situ mPA6115 mice; group 4 = in situ mPA6115 mice treated with anti-Gal 9 mAb; group 5 = gemcitabine/Abraxane treated in situ mPA6115 mice; and group 6 = in situ mPA6115 mice treated with anti-Gal 9 mAb and gemcitabine/Abraxane.
Fig. 3 includes a graph of average body weight of each treated group measured twice weekly during the study, with group 1 = untreated in situ mPA6115 mice; group 2 = chemical vehicle control, saline treated in situ mPA6115 mice; group 3 = isotype IgG1 mice treated in situ mPA6115 mice; group 4 = in situ mPA6115 mice treated with anti-Gal 9 mAb; group 5 = gemcitabine/Abraxane treated in situ mPA6115 mice; and group 6 = in situ mPA6115 mice treated with anti-Gal 9 mAb and gemcitabine/Abraxane.
Fig. 4 is a schematic diagram depicting an exemplary study protocol. CRM: re-evaluation method; RP2D: recommended phase 2 dose; PK: pharmacokinetics; PD: pharmacodynamics; PDAC: pancreatic ductal adenocarcinoma; CRC: colorectal cancer; CCA: bile duct cancer; TBD: to be determined.
FIG. 5 includes graphs showing the effect of G2.9-17 on TGF- β1 secretion measurements in an exemplary healthy human donor whole blood. TGF- β1 is released from donor cryopreserved macrophages incubated in the presence of the M2 polarization mixture. IgG4 isotype is the negative control antibody. Data represent mean + SEM of triplicate measurements. Significance was determined by two-way ANOVA using the Dunnett multiple comparison test. * p <0.05
FIG. 6 includes graphs showing the effect of G2.9-17 on IL-10 secretion in whole blood of an exemplary healthy human donor. IL-10 was released from donor cryopreserved macrophages incubated in the presence of M2 polarization mixtures (IL-4/IL-13 or Gal-9). IgG4 isotype is the negative control antibody. Data represent mean (±sem) of triplicate. Significance was determined by two-way ANOVA using Tukey multiple comparison test. * P <0.05.
Detailed Description
Provided herein are methods of treating cancer using anti-galectin-9 antibodies (e.g., G9.2-17) in combination with chemotherapeutic agents such as gemcitabine and paclitaxel (e.g., protein-bound paclitaxel, such as nanoparticulate albumin-bound paclitaxel, e.g.,) To treat solid tumors, such as Pancreatic Ductal Adenocarcinoma (PDAC). In some embodiments, the solid tumor is metastatic. In some embodiments, the methods disclosed herein provide specific dosages and/or dosing regimens. In some cases, the methods disclosed herein target a particular patient population, e.g., a patient who has undergone prior treatment and who has shown disease progression through prior treatment, or a patient who is resistant (from the head or acquired) to prior treatment.
In some embodiments, the methods disclosed herein provide specific dosages and/or dosing regimens of an anti-Gal-9 antibody disclosed herein (e.g., G9.2-17 (IgG 4)) in combination with a chemotherapeutic agent also disclosed herein (e.g., gemcitabine and paclitaxel), e.g., an antibody of 0.2mg/kg to 16mg/kg once per week to once per 4 weeks (e.g., 0.2mg/kg, 0.63mg/kg, 2mg/kg, 6.3mg/kg, 10mg/kg, or 16mg/kg once per week or once per two weeks). In some examples, the dosing regimen of an anti-Gal 9 antibody, such as G9.2-17 (IgG 4), can be 10mg/kg or 16mg/kg once a week. In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week.
Alternatively, an anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG 4), may be administered to a subject once per week to once every 4 weeks (e.g., once per week or once every two weeks) at a stationary dose, for example, about 650mg to about 1120mg (e.g., about 650-700mg or about 1040-1120 mg). G9.2-17 (IgG 4) was found to have unexpectedly rapid clearance in human subjects compared to conventional antibody therapeutics. Thus, a treatment regimen comprising a weekly dosing schedule was developed to ensure systemic exposure levels of anti-Gal-9 antibodies that achieved therapeutic effects.
Galectin-9 (a tandem repeat lectin) is a β -galactoside binding protein that has been shown to play a role in regulating cell-cell and cell-matrix interactions. It has been found to be strongly overexpressed in hodgkin's tissues and other pathological conditions. In some cases, it was also found to circulate in the Tumor Microenvironment (TME).
Galectin-9 interacts with Dectin-1 (an innate immune receptor highly expressed on macrophages in PDAC and on cancer cells) (Daley et al, nat Med.2017;23 (5): 556-6). Regardless of the source of galectin-9, disrupting its interaction with Dectin-1 has been shown to cause the binding of CD4 + And CD8 + Cells are reprogrammed as an integral mediator of anti-tumor immunity. Thus, galectin-9 serves as a valuable therapeutic target for blocking the signaling mediated by Dectin-1. Thus, in some embodiments, an anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and Dectin-1.
Galectin-9 can also interact with TIM-3, a type I cell surface glycoprotein, expressed on the surface of leukemic stem cells of all kinds of acute myeloid leukemia, except M3 (acute promyelocytic leukemia), but not in normal human Hematopoietic Stem Cells (HSCs). TIM-3 signaling resulting from galectin-9 ligation has been found to have pleiotropic effects on immune cells, inducing apoptosis of Th1 cells (Zhu et al, nat immunol.,2005, 6:1245-1252) and stimulating secretion of tumor necrosis factor- α (TNF- α), causing monocytes to mature into dendritic cells, resulting in inflammation caused by innate immunity (Kuchroo et al, nat Rev immunol.,2008, 8:577-580). Additional galectin-9/TIM-3 signaling has been found to co-activate NF-. Kappa.B and β -catenin signaling (two pathways that promote LSC self-renewal) (Kikushige et al, cell Stem Cell,2015,17 (3): 341-352). Anti-galectin-9 antibodies that interfere with galectin-9/TIM-3 binding may have therapeutic effects, particularly on leukemia and other hematological malignancies. Thus, in some embodiments, an anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and TIM-3.
Furthermore, galectin-9 interacts with CD206 (a mannose receptor, highly expressed on M2 polarized macrophages, thereby promoting tumor survival) (Enning ga et al, J Pathol.2018, month 8; 245 (4): 468-477). Tumor-associated macrophages expressing CD206 are mediators of tumor immunosuppression, angiogenesis, metastasis and recurrence (see, e.g., scoodeller et al, sci Rep.2017, month 11, 7; 7 (1): 14655, and references therein). In particular, M1 (also known as classical activated macrophages) is triggered by Th 1-associated cytokines and bacterial products, expresses high levels of IL-12, and has a tumoricidal effect. In contrast, M2 (a so-called surrogate activated macrophage) is activated by Th 2-associated factors, expresses high levels of anti-inflammatory cytokines such as IL-10, and promotes tumor progression (Biswas and Mantovani; nat immunol.10 month 2010; 11 (10): 889-96). The pro-neoplastic effects of M2 include promoting angiogenesis, promoting invasion and metastasis, and protecting tumor cells from chemotherapy-induced apoptosis (Hu et al, tumour biol.2015, 12; 36 (12): 9119-9126, and references therein). Tumor-associated macrophages are believed to have an M2-like phenotype and have a tumorigenic effect. Galectin-9 has been shown to mediate differentiation of bone marrow cells to the M2 phenotype (Enning et al, melanoma Res.2016, 10; 26 (5): 429-41). Galectin-9 binding to CD206 potentially results in reprogramming TAM to M2 phenotype, similar to what was shown previously for Dectin. Without wishing to be bound by theory, blocking the interaction of galectin-9 with CD206 may provide a mechanism by which anti-galectin-9 antibodies (e.g., G9.2-17 antibodies) may be therapeutically beneficial. Thus, in some embodiments, an anti-galectin-9 antibody described herein disrupts the interaction between galectin-9 and CD 206.
Galectin-9 has also been shown to interact with Protein Disulfide Isomerase (PDI) and 4-1BB (Bi S et al Proc Natl Acad Sci U S A.2011;108 (26): 10650-5; madireddi et al J Exp Med.2014;211 (7): 1433-48).
Anti-galectin-9 antibodies can serve as therapeutic agents for treating diseases associated with galectin-9 (e.g., those in which galectin-9 signaling plays a role). Without being bound by theory, anti-galectin-9 antibodies may block galectin-9 mediated signaling pathways. For example, an antibody may interfere with the interaction between galectin-9 and its binding partner (e.g., dectin-1, TIM-3, or CD 206), thereby blocking signaling triggered by galectin-9/ligand interactions. Alternatively, or in addition, anti-galectin-9 antibodies may also exert their therapeutic effect by inducing blocking and/or cytotoxicity, e.g. ADCC, CDC or ADCP against galectin-9 expressing pathological cells. Pathological cells refer to cells that directly or indirectly contribute to the occurrence and/or progression of a disease. See, e.g., WO2019/084553, WO2020/198390, WO2020/0223702 and WO2021022256, the relevant disclosures of each of which are incorporated by reference for the subject matter and purposes cited herein.
The anti-galectin-9 antibodies disclosed herein are capable of inhibiting galectin-9 mediated signaling (e.g., a galectin-9/Dectin-1 or galectin-9/Tim-3 mediated signaling pathway) or eliminating pathological cells expressing galectin-9 via, for example, ADCC. Thus, the anti-galectin-9 antibodies described herein can be used to inhibit any galectin-9 signaling and/or eliminate galectin-9 positive pathological cells, thereby being beneficial for treating galectin-9 related diseases. See, e.g., WO2019/084553, PCT/US2020/024767, and PCT/US2020/031181, the relevant disclosures of each of which are incorporated by reference for the purposes and subject matter cited herein.
As reported herein, combination therapy of representative anti-Gal 9 antibodies (G9.2-17 such as G9.2-17 (IgG 4)) and chemotherapeutic agents (gemcitabine and nab-paclitaxel) successfully prolonged survival in animal models as disclosed herein. Synergy of representative anti-Gal 9 antibodies with gemcitabine and nab-paclitaxel on survival time was observed in animal models. These results demonstrate that the anti-tumor methods disclosed herein, involving a combination of an anti-galectin-9 antibody and a chemotherapeutic agent such as those disclosed herein, will achieve better therapeutic efficacy against a target solid tumor than either the antibody or the chemotherapeutic agent alone.
Thus, described herein are therapeutic uses of anti-galectin-9 antibodies and chemotherapeutic agents as disclosed herein for the treatment of certain solid tumors.
Antibodies that bind to galectin-9
The present disclosure provides anti-Gal-9 antibodies G9.2-17 and functional variants thereof for use in the methods of treatment disclosed herein.
Antibodies (used interchangeably in plural form) are immunoglobulin molecules capable of specifically binding to a target (such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody", e.g., anti-galectin-9 antibody, includes not only intact (e.g., full length) polyclonal or monoclonal antibodies, but also antigen binding fragments thereof (such as Fab, fab ', F (ab') 2, fv), single chains (scFv), mutants thereof, fusion proteins comprising an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. Antibodies (e.g., anti-galectin-9 antibodies) include antibodies of any class, such as IgD, igE, igG, igA or IgM (or subclass thereof), and the antibodies need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the antibody heavy chain constant domain. Immunoglobulins fall into five main categories: igA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
Typical antibody molecules comprise a heavy chain variable region (V H ) And a light chain variable region (V L ) They are generally involved in antigen binding. V (V) H And V L The regions may be further subdivided into regions of high variability, also known as "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, known as "framework regions" ("FR"). Each V H And V L Typically consisting of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The scope of framework regions and CDRs may be precisely identified using methods known in the art (e.g., by Kabat definition, chothia definition, abM definition, EU definition, "Contact" numbering scheme, "IMGT" numbering scheme, "AHo" numbering scheme, and/or Contact definition, all of which are well known in the art). See, e.g., kabat, e.a. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Pat. No. of Health and Human Services, NIH Publication No.91-3242; chothia et al, (1989) Nature 342:877; chothia, C.et al (1987) J.mol.biol.196:901-917; al-lazikani et Al (1997) J.molecular.biol.273:927-948; edelman et al Proc Natl Acad Sci U S a.1969, month 5; 63 (1) 78-85; and Almagro, J.mol. Recognit.17:132-143 (2004); macCallum et al, J.mol. Biol.262:732-745 (1996), lefranc M P et al, dev Comp Immunol, month 1 2003; 27 (1) 55-77; and honeygger a and plurkthun a, J Mol Biol, 6/8/2001; 309 (3):657-70. See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).
In some embodiments, the anti-galectin-9 antibodies described herein are full length antibodies that contain two heavy chains and two light chains, each comprising a variable domain and a constant domain. Alternatively, the anti-galectin-9 antibody may be an antigen-binding fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of a full-length antibody include (i) Fab fragments, consisting of V L 、V H 、C L And C H 1 domain; (ii) F (ab') 2 Fragments comprising a disulfide group in the hinge regionA bivalent fragment of two Fab fragments linked by a bond; (iii) From V H And C H 1 domain-composed Fd fragment; (iv) V by antibody single arm L And V H Fv fragments consisting of domains; (v) dAb fragment (Ward et al, (1989) Nature 341:544-546), which is defined by V H Domain composition; and (vi) an isolated Complementarity Determining Region (CDR) that retains function. Furthermore, although the two domains V of the Fv fragment L And V H Encoded by different genes, but they can be joined by synthetic linkers using recombinant methods, enabling them to be made into individual protein chains, where V L And V H The pairing of regions forms monovalent molecules, known as single chain Fv (scFv). See, e.g., bird et al (1988) Science 242:423-426; and Huston et al (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883.
Any of the antibodies described herein, e.g., an anti-galectin-9 antibody, may be monoclonal or polyclonal. "monoclonal antibody" refers to a homogeneous population of antibodies and "polyclonal antibody" refers to a heterogeneous population of antibodies. These two terms do not limit the source of the antibody or the manner in which it is made.
Reference antibodies G9.2-17 refer to antibodies capable of binding human galectin-9 and comprise the heavy chain variable region of SEQ ID NO. 7 and the light chain variable domain of SEQ ID NO. 8, both of which are provided below. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is a G9.2-17 antibody. In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein is an antibody having the same heavy chain Complementarity Determining Regions (CDRs) as the reference antibodies G9.2-17 and/or the same light chain complementarity determining regions as the reference antibodies G9.2-17. With the same V H And/or V L Both antibodies of a CDR means that their CDRs are identical when determined by the same method (e.g., kabat method, chothia method, abM method, contact method or IMGT method as known in the art, see e.g., bioif. Org. Uk/abs /).
The heavy and light chain CDRs of reference antibodies G9.2-17 are provided in table 1 below (determined using the Kabat method):
TABLE 1 heavy and light chain CDRs of G9.2-17
In some examples, an anti-galectin-9 antibody for use in the methods disclosed herein may comprise (according to the Kabat protocol) heavy chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 4, heavy chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 5, and heavy chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 6, and/or may comprise light chain complementarity determining region 1 (CDR 1) as shown in SEQ ID NO. 1, light chain complementarity determining region 2 (CDR 2) as shown in SEQ ID NO. 2, and light chain complementarity determining region 3 (CDR 3) as shown in SEQ ID NO. 3. The anti-galectin-9 antibody (including reference antibody G9.2-17) may be in any form as disclosed herein, such as a full length antibody or Fab. The term "G9.2-17 (Ig 4)" as used herein refers to the G9.2-17 antibody as an IgG4 molecule. Also, the term "G9.2-17 (Fab)" refers to the G9.2-17 antibody as a Fab molecule.
In some embodiments, an anti-galectin-9 antibody or binding portion thereof comprises heavy and light chain variable regions, wherein the light chain variable region CDR1, CDR2, and CDR3 amino acid sequences have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increments therein) sequence identity to the light chain variable region CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs 1, 2, and 3, respectively. In some embodiments, an anti-galectin-9 antibody or binding portion thereof comprises heavy and light chain variable regions, wherein the heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and any increments therein) sequence identity to the heavy chain variable region CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs 4, 5, and 6, respectively.
Additional galectin-9 antibodies (e.g., binding to CRD1 and/or CRD2 regions of galectin-9) are described in commonly owned, co-pending U.S. patent application 16/173,970 and commonly owned, co-pending international patent applications PCT/US18/58028 and PCT/US2020/024767, the contents of each of which are incorporated herein by reference in their entirety.
In some embodiments, an anti-galectin-9 antibody disclosed herein comprises light chain CDRs that correspond to V of reference antibody G9.2-17 L CDRs have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity compared to either alone or together. Alternatively or additionally, in some embodiments, the anti-galectin-9 antibody comprises a heavy chain CDR that is identical to the corresponding V of the reference antibody G9.2-17 H CDRs have at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity compared to either alone or together.
The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68,1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77,1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al J.mol.biol.215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the protein molecules of the present invention. When a gap exists between the two sequences, gap BLAST can be used, as described in Altschul et al, nucleic Acids Res.25 (17): 3389-3402, 1997. When using BLAST and gapped BLAST programs, default parameters for the respective programs (e.g., XBLAST and NBLAST) can be used.
In other embodiments, an anti-galectin-9 antibody described herein contains a V comprising HC CDR1, HC CDR2, and HC CDR3 H Together they comprise up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variations, including additions, deletions and/or substitutions) of HC CDR1, HC CDR2 and HC CDR3 relative to a reference antibody G9.2-17. Alternatively or additionally, in some embodiments, an anti-galectin-9 antibody described herein comprises V H The VH comprises LC CDR1, LC CDR2 and LC CDR3 that together contain up to 8 amino acid residue variations (8, 7, 6, 5, 4, 3, 2 or 1 variations, including additions, deletions and/or substitutions) relative to LC CDR1, LC CDR2 and LC CDR3 of reference antibody G9.2-17.
In one example, the amino acid residue variation is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which they are made. Variants can be made according to methods known to those of ordinary skill in the art for altering the sequence of a polypeptide, such as found in references compiling such methods, for example Molecular Cloning: A Laboratory Manual, J.Sambrook et al, second edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, new York,1989, or Current Protocols in Molecular Biology, F.M. Ausubel et al, john Wiley & Sons, inc., new York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
In some embodiments, an anti-galectin-9 antibody disclosed herein having heavy chain CDRs disclosed herein contains a polypeptide derived from germline V H Framework regions of fragment subclasses. Such a line V H Regions are well known in the art. See, for example, IMGT database (www.imgt.org) or www.vbase2.org/vbstat. Examples include IGHV1 subfamilies (e.g., IGHV1-2, IGHV1-3, IGHV1-8, IGHV1-18, IGHV1-24, IGHV1-45, IGHV1-46, IGHV1-58, and IGHV 1-69), IGHV2 subfamilies (e.g., IGHV2-5, IGHV2-26, and IGHV 2-70), IGHV3 subfamilies (e.g., IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-20, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-33, IGHV3-43, IGHV3-48, IGHV3-49, IGHV3-53, IGHV3-64, IGHV3-66, IGHV3-72, and IGHV3-73, IGHV 3-74), IGHV4 subfamilies (e.g., IGHV4-4, IGHV4-28, IGHV4-31, IGHV4-34, IGHV4-4, and IGHV4-6, or IGHV4-6, e.g., and IGHV 1-7.
Alternatively or additionally, in some embodiments, an anti-galectin-9 antibody having light chain CDRs as disclosed herein contains a framework region derived from a germline vκ fragment. Examples include IGKV1 frames (e.g., IGKV1-05, IGKV1-12, IGKV1-27, IGKV1-33, or IGKV 1-39), IGKV2 frames (e.g., IGKV 2-28), IGKV3 frames (e.g., IGKV3-11, IGKV3-15, or IGKV 3-20), and IGKV4 frames (e.g., IGKV 4-1). In other cases, the anti-galectin-9 antibody comprises a light chain variable region containing a framework derived from a germline V lambda fragment. Examples include IGλ1 frames (e.g., IGλV1-36, IGλV1-40, IGλV1-44, IGλV1-47, IGλV 1-51), IGλ2 frames (e.g., IGλV2-8, IGλV2-11, IGλV2-14, IGλV2-18, IGλV 2-23), IGλ3 frames (e.g., IGλV3-1, IGλV3-9, IGλV3-10, IGλV3-12, IGλV3-16, IGλV3-19, IGλV3-21, IGλV3-25, IGλV 3-27), IGλ4 frames (e.g., IGλV4-3, IGλV4-60, IGλV 4-69), λ5 frames (e.g., IGλV5-39, IGλV 5-45), IGλ6 frames (e.g., λV 6-57), IGλ7 frames (e.g., IGλV7-43, λV 7-8, IGλV 7-43), IGλV8 (e.g., IGλV 9-9, or IGλ9-10, e.g., IGλV 8-9).
In some embodiments, the anti-galectin-9 antibody used in the methods disclosed herein may be a heavy chain antibody having the same variable region (V H ) And/or identical light chain variable regions (V L ) The antibody of V H And V L The region amino acid sequence is provided below:
V H ：
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSS(SEQ ID NO:7)
V L ：
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIKR(SEQ ID NO:8)
in some embodiments, the anti-galectin-9 antibody has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to the heavy chain variable region of SEQ ID NO: 7. Alternatively or additionally, the anti-galectin-9 antibody has at least 80% sequence identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to the light chain variable region of SEQ ID NO: 8.
In some cases, the anti-galectin-9 antibody disclosed herein is a functional variant of reference antibody G9.2-17. Functional variants may be similar in structure to a reference antibody (e.g., comprising a limited number of amino acid residue variations in one or more of the heavy and/or light chain CDRs of G9.2-17 as disclosed herein, or sequence identity to the heavy and/or light chain CDRs of G9.2-17 or VH and/or VL of G9.2-17 as disclosed herein), with substantially similar binding affinity to human galectin-9 (e.g., with a similar KD value).
In some embodiments, an anti-galectin-9 antibody as described herein can bind to galectin-9 and inhibit its activity by at least 20% (e.g., 31%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Apparent inhibition constant (Ki) app Or K i,app ) Is related to the concentration of inhibitor required to reduce the enzyme activity and is independent of the enzyme concentration. The inhibitory activity of the anti-galectin-9 antibodies described herein can be determined by conventional methods known in the art.
Antibody K i , app The value may be determined by the following procedure: measuring the inhibition of the extent of the reaction (e.g., enzyme activity) by different concentrations of antibody; the change in the pseudo first order rate constant (v) as a function of inhibitor concentration is fitted to the modified Morrison equation (equation 1) to yield an estimate of the apparent Ki value. Ki for competitive inhibitors app Can be extracted from K i , app The y-intercept of the linear regression analysis with the substrate concentration plot was obtained.
Wherein A is equal to v o E, initial rate of enzymatic reaction in the absence of inhibitor (I) (v o ) Divided by the total enzyme concentration (E). In some embodiments, an anti-galectin-9 antibody described herein has a Ki of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5pM or less for a target antigen or epitope app Values. In some embodiments, the anti-galectin-9 antibody has a lower Ki for the first target (e.g., CRD2 of galectin-9) relative to the second target (e.g., CRD1 of galectin-9) app 。Ki app May be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 Multiple times. In some examples, the anti-galectin-9 antibody inhibits a first antigen (e.g., a first protein or mimetic thereof in a first conformation) more than a second antigen (e.g., the same first protein or mimetic thereof in a second conformation; or a second protein). In some embodiments, any of the anti-galectin-9 antibodies is further affinity matured to reduce the Ki of the antibody to the target antigen or epitope thereof app 。
In some embodiments, the anti-galectin-9 antibody inhibits Dectin-1 signaling in tumor-infiltrating immune cells such as macrophages, for example. In some embodiments, an anti-galectin-9 antibody inhibits galectin-9 triggered Dectin-1 signaling by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays. Alternatively or additionally, the anti-galectin-9 antibody inhibits galectin-9-initiated T cell immunoglobulin mucin-3 (TIM-3) signaling. In some embodiments, an anti-galectin-9 antibody inhibits T cell immunoglobulin mucin-3 (TIM-3) signaling, e.g., in tumor infiltrating immune cells, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein), e.g., in some embodiments. Such inhibition activity may be determined by conventional methods of assay such as conventional assays.
In some embodiments, the anti-galectin-9 antibody inhibits CD206 signaling in, for example, tumor infiltrating immune cells. In some embodiments, an anti-galectin-9 antibody inhibits CD206 signaling triggered by galectin-9 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays. In some embodiments, the anti-galectin-9 antibody blocks or prevents binding of galectin-9 to CD206 by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays.
In some embodiments, the anti-galectin-9 antibody induces cytotoxicity, such as ADCC, in a target cell expressing galectin-9, e.g., wherein the target cell is a cancer cell or an immunosuppressive immune cell. In some embodiments, an anti-galectin-9 antibody induces apoptosis in an immune cell, such as a T cell or a cancer cell, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibition activity may be determined by conventional methods of assay such as conventional assays. In some embodiments, any of the anti-galectin-9 antibodies described herein induces cytotoxicity, such as Complement Dependent Cytotoxicity (CDC), against target cells expressing galectin-9.
Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action of antibodies through phagocytosis to mediate some or all of their actions. In this case, the antibody mediates uptake of the specific antigen by the antigen presenting cells. ADCP can be mediated by monocytes, macrophages, neutrophils and dendritic cells through fcyriia, fcyri and fcyriiia, with fcyriia on macrophages (CD 32 a) representing the primary pathway.
In some embodiments, the anti-galectin-9 antibody induces cell phagocytosis (ADCP) of target cells (e.g., cancer cells or immunosuppressive immune cells that express galectin-9). In some embodiments, the anti-galectin-9 antibody increases phagocytosis of a target cell, e.g., a cancer cell or an immunosuppressive immune cell, by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).
In some embodiments, an anti-galectin-9 antibody described herein induces cytotoxicity, such as Complement Dependent Cytotoxicity (CDC), against a target cell, e.g., a cancer cell or an immunosuppressive immune cell. In some embodiments, an anti-galectin-9 antibody increases CDC against a target cell by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).
In some embodiments, the anti-galectin-9 antibody induces T cell activation, e.g., in tumor infiltrating T cells, i.e., directly or indirectly inhibits galectin-9 mediated inhibition of T cell activation. In some embodiments, an anti-galectin-9 antibody increases T cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). T cell activation may be determined by conventional methods, such as assays (e.g., measuring CD44, tnfa, ifnγ, and/or PD-1). In some embodiments, the anti-galectin-9 antibody increases cd4+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, an anti-galectin antibody induces CD44 expression in cd4+ cells. In some embodiments, the anti-galectin-9 antibody increases CD44 expression in cd4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any delta therein). In a non-limiting example, an anti-galectin antibody induces ifnγ expression in cd4+ cells. In some embodiments, the anti-galectin-9 antibody increases ifnγ expression in cd4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any delta therein). In a non-limiting example, an anti-galectin antibody induces tnfα expression in cd4+ cells. In some embodiments, an anti-galectin-9 antibody increases tnfα expression in cd4+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).
In some embodiments, the anti-galectin-9 antibody increases cd8+ cell activation by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increments therein). In a non-limiting example, an anti-galectin antibody induces CD44 expression in cd8+ cells. In some embodiments, the anti-galectin-9 antibody increases CD44 expression in cd8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increase therein). In a non-limiting example, an anti-galectin antibody induces ifnγ expression in cd8+ cells. In some embodiments, the anti-galectin-9 antibody increases ifnγ expression in cd8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein). In a non-limiting example, an anti-galectin antibody induces tnfα expression in cd8+ cells. In some embodiments, an anti-galectin-9 antibody increases tnfα expression in cd8+ cells by at least 30% (e.g., 31%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, including any increment therein).
In some implementationsIn embodiments, an anti-galectin-9 antibody as described herein has suitable binding affinity for a target antigen (e.g., galectin-9) or an epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A 。K A Is the dissociation constant (K) D ) Is the inverse of (c). The anti-galectin-9 antibodies described herein may have at least 10 for a target antigen or epitope -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 M or lower binding affinity (K D ). The increase in binding affinity corresponds to K D And (3) lowering. Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescent assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150mM NaCl, 0.005% (v/v) surfactant P20).
These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. Under certain conditions, the fractional concentration of bound binding protein ([ binding ]/[ total ]) is generally related to the concentration of total target protein ([ target ]) by the following equation:
binding/total target/Kd target
However, it is not always necessary to accurately determine K A Because quantitative measurements of affinity are sometimes obtained (e.g., determined using methods such as ELISA or FACS analysis, with K) A Proportionality, therefore, may be used for comparison, such as determining if the higher affinity is higher, e.g., by a factor of 2), obtaining qualitative measurements of affinity, or obtaining inferences of affinity (e.g., by activity in a functional assay (e.g., in vitro or in vivo assay) are sufficient. In some cases, the in vitro binding assay is indicative of in vivo activity. In other cases, an in vitro binding assay is not necessarily indicative of in vivo activity. In some cases tight binding is beneficial, but in other cases tight binding is not ideal in vivo, and antibodies with lower binding affinity are more ideal.
In some embodiments, the heavy chain of any of the anti-galectin-9 antibodies as described herein further comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, for example human, mouse, rat or rabbit. In a specific example, the heavy chain constant region is from human IgG (gamma heavy chain) of any IgG subfamily as described herein.
In some embodiments, the heavy chain constant regions of antibodies described herein comprise a single domain (e.g., CH1, CH2, or CH 3) or a combination of any single domain of the constant regions (e.g., SEQ ID NOS: 10, 12-14, and 21). In some embodiments, the light chain constant regions of antibodies described herein comprise a single domain of constant region (e.g., CL). Exemplary light and heavy chain sequences are listed below. Exemplary light and heavy chain sequences are listed below. The hIgG1 LALA sequence includes two mutations (L234A and L235A (EU numbering) that inhibit FcgR binding) and a P329G mutation (EU numbering) to eliminate complement C1q binding, thereby eliminating all immune effector functions. The hIgG4 Fab arm crossover mutant sequence includes a mutation that inhibits Fab arm crossover (S228P; EU numbering). IL2 signal sequence (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 9) may be located at the N-terminus of the variable region. It is used in expression vectors, which are cleaved during secretion and therefore not in mature antibody molecules. The heavy chain of the mature protein (post-secretion) starts with "EVQ" and the light chain with "DIM". The amino acid sequences of exemplary heavy chain constant regions are provided below:
hIgG1 heavy chain constant region (SEQ ID NO: 10)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
hIgG1 LALA heavy chain constant region (SEQ ID NO: 12)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
hIgG4 heavy chain constant region (SEQ ID NO: 13)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*
hIgG4 heavy chain constant region (SEQ ID NO: 20)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
hIgG4 mutant heavy chain constant region (SEQ ID NO: 14)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*
hIgG4 mutant heavy chain constant region (SEQ ID NO: 21)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
In some cases, the heavy chain constant region in an anti-galectin-9 antibody (e.g., G9.2-17) disclosed herein can remove a C-terminal lysine (K) residue for manufacturing purposes, for example. Corresponding amino acid sequences to those without terminal K residues are provided below:
hIgG1 heavy chain constant region without C-terminal lysine (SEQ ID NO: 24)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*
hIgG1 LALA heavy chain constant region without C-terminal lysine (SEQ ID NO: 25)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*
hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 26)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*
hIgG4 heavy chain constant region without C-terminal lysine (SEQ ID NO: 27)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*
hIgG4 mut heavy chain constant region without C-terminal lysine (SEQ ID NO: 28)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*
hIgG4 mut heavy chain constant region without C-terminal lysine (SEQ ID NO: 29)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*
In some embodiments, an anti-galectin-9 antibody having any one of the above heavy chain constant regions is paired with a light chain having the following light chain constant regions:
light chain constant region (SEQ ID NO: 11)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Exemplary full-length anti-galectin-9 antibodies are provided below:
G9.2-17hIgG1 heavy chain (SEQ ID NO: 16)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
G9.2-17hIgG1 heavy chain without C-terminal lysine residue (SEQ ID NO: 30)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*
G9.2-17hIgG1 LALA heavy chain (SEQ ID NO: 17)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
G9.2-17hIgG1 LALA heavy chain without C-terminal lysine residue (SEQ ID NO: 31)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG*
G9.2-17hIgG4 heavy chain (SEQ ID NO: 18)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*
G9.2-17hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 32)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*
G9.2-17hIgG4 heavy chain (SEQ ID NO: 22)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
G9.2-17hIgG4 heavy chain without C-terminal lysine residue (SEQ ID NO: 33)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*
G9.2-17hIgG4 Fab arm exchange mutant heavy chain (SEQ ID NO: 19)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*
G9.2-17hIgG4 Fab arm exchange mut heavy chain without C-terminal lysine residue (SEQ ID NO: 34)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG*
G9.2-17hIgG4 Fab arm exchange mutant heavy chain (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
G9.2-17hIgG4 Fab arm exchange mut heavy chain without C-terminal lysine residue (SEQ ID NO: 35)
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSSSIHWVRQAPGKGLEWVAYISSSSGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYWSYPSWWPYRGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*
Any of the heavy chains described above may be paired with a light chain as shown below (SEQ ID NO: 15):
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQSSTDPITFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*
in some embodiments, an anti-galectin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO 10. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region comprising SEQ ID NO. 13. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO. 10.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO. 13. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO. 13. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO. 13.
In some embodiments, the constant region is from human IgG4. In one embodiment, an anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO. 20. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO. 20. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO. 20.
In any of these embodiments, the anti-galectin-9 antibody comprises a light chain constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID No. 11. In some embodiments, the anti-galectin-9 antibody comprises a light chain constant region comprising SEQ ID NO. 11. In some embodiments, the anti-galectin-9 antibody comprises a light chain constant region consisting of SEQ ID NO. 11.
In some embodiments, the IgG is a mutant with minimal Fc receptor binding. In one example, the constant region is from human IgG1 LALA. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO. 12. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region comprising SEQ ID NO. 12. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG1 constant region consisting of SEQ ID NO. 12.
In some embodiments, the anti-galectin-9 antibody comprises a modified constant region. In some embodiments, the anti-galectin-9 antibody comprises an immunologically inert modified constant region, e.g., does not trigger complement-mediated lysis, or does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC activity may be assessed using the methods disclosed in U.S. patent No. 5,500,362. In other embodiments, the constant regions are modified as described in Eur.J.Immunol. (1999) 29:2613-2624, PCT application No. PCT/GB99/01441 and/or British patent application No. 9809951.8. In some embodiments, the IgG4 constant region is a mutant with reduced heavy chain exchange. In some embodiments, the constant region is from human IgG4 Fab arm swap mutant S228P.
In one embodiment, the constant region of an anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID NO. 14. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO. 14. In one embodiment, the constant region of the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO. 14.
In one embodiment, an anti-galectin-9 antibody comprises a heavy chain IgG4 constant region having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID NO. 21. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region comprising SEQ ID NO. 21. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain IgG4 constant region consisting of SEQ ID NO. 21.
In some embodiments, the light chain of the anti-galectin-9 antibody has a chain corresponding to SEQ ID NO. 15; and the amino acid sequence of an exemplary heavy chain corresponds to SEQ ID NO. 10 (hIgG 1); 12 (hIgG 1 LALA); 13 (hIgG 4); 20 (hIgG 4); 14 (hIgG 4 mut); and 21 (hIgG 4 mut).
In some embodiments, the anti-galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO. 15. In some embodiments, the anti-galectin-9 antibody has a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NOS 16-19, 22, and 23. In some embodiments, the anti-galectin-9 antibody has a light chain comprising, consisting essentially of, or consisting of SEQ ID NO. 15 and a heavy chain comprising, consisting essentially of, or consisting of any one of the sequences selected from the group consisting of SEQ ID NO. 16-19. In some embodiments, the anti-galectin-9 antibody has a light chain comprising SEQ ID NO. 15 and a heavy chain comprising any one of the sequences selected from the group consisting of SEQ ID NO. 16-19, 22 and 23. In some embodiments, the anti-galectin-9 antibody has a light chain consisting essentially of SEQ ID NO. 15 and a heavy chain consisting essentially of any one of the sequences selected from the group consisting of SEQ ID NO. 16-19, 22 and 23. In some embodiments, the anti-galectin-9 antibody has a light chain consisting of SEQ ID NO. 15 and a heavy chain consisting of any one of the sequences selected from the group consisting of SEQ ID NO. 16-19, 22 and 23. In a specific embodiment, the anti-galectin-9 antibody has a light chain consisting essentially of SEQ ID NO. 15 and a heavy chain consisting essentially of SEQ ID NO. 19. In another specific embodiment, the anti-galectin-9 antibody has a light chain consisting essentially of SEQ ID NO. 15 and a heavy chain consisting essentially of SEQ ID NO. 20.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID No. 16. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO. 16. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO. 16.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID No. 17. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO. 17. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO. 17.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID No. 18. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO. 18. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO. 18.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID No. 22. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO. 22. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO. 22.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID No. 19. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO. 19. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO. 19.
In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% and any increments therein) sequence identity to SEQ ID No. 23. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence comprising SEQ ID NO. 23. In one embodiment, the anti-galectin-9 antibody comprises a heavy chain sequence consisting of SEQ ID NO. 23.
In any of these embodiments, the anti-galectin-9 antibody comprises a light chain sequence having at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and any increments therein) sequence identity to SEQ ID No. 15. In some embodiments, the anti-galectin-9 antibody comprises a light chain sequence comprising SEQ ID NO. 15. In some embodiments, the anti-galectin-9 antibody comprises a light chain sequence consisting of SEQ ID NO. 15.
In a specific example, an anti-galectin-9 antibody used in the methods of treatment disclosed herein has a heavy chain of SEQ ID NO. 19 and a light chain of SEQ ID NO. 15. In some embodiments, the anti-galectin-9 antibody used in the methods of treatment disclosed herein is G9.2-17IgG4. In some examples, such anti-galectin-9 antibodies do not have a C-terminal lysine residue in their heavy chain.
Preparation of anti-galectin-9 antibodies
Antibodies capable of binding galectin-9 as described herein may be prepared by any method known in the art, including but not limited to recombinant techniques. An example is provided below.
Nucleic acids encoding the heavy and light chains of an anti-galectin-9 antibody as described herein can be cloned into an expression vector, each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains may be operably linked to a single promoter such that both the heavy and light chains are expressed from the same promoter. If necessary, an Internal Ribosome Entry Site (IRES) can be inserted between the heavy and light chain coding sequences.
In some examples, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which may be introduced into the same or different cells. When the two chains are expressed in different cells, each of them may be isolated from the host cell in which it is expressed, and the isolated heavy and light chains may be mixed and incubated under suitable conditions that allow the formation of antibodies.
Generally, nucleic acid sequences encoding one or all of the chains of an antibody can be cloned into a suitable expression vector operably linked to a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector may be contacted with a restriction enzyme under suitable conditions to produce complementary ends on each molecule that can be paired with each other and linked together with a ligase. Alternatively, a synthetic nucleic acid linker may be attached to the end of the gene. These synthetic linkers contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector/promoter will depend on the type of host cell used to produce the antibody.
A variety of promoters may be used to express the antibodies described herein, including but not limited to the Cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma (Rous sarcoma) viral LTR, HIV-LTR, HTLV-1LTR, simian Virus 40 (SV 40) early promoter, E.coli) lac UV5 promoter, and herpes simplex tk virus promoter.
Regulatable promoters may also be used. Such regulatable promoters include those that use a lac repressor from E.coli as a transcription regulator to regulate transcription of mammalian Cell promoters harboring the lac operon [ Brown, M.et al, cell,49:603-612 (1987) ], those that use a tetracycline repressor (tetR) [ Gossen, M.and Bujard, H., proc.Natl.Acad.Sci.USA 89:5547-5551 (1992); yao, F. Et al, human Gene Therapy,9:1939-1950 (1998); sockelt, P.et al, proc.Natl. Acad.Sci.USA,92:6522-6526 (1995) ]. Other systems include FK506 dimer, VP16 or p65 using estradiol, RU486, diphenol Le Zai ketone (diphenol murislerone) or rapamycin. Inducible systems are available from Invitrogen, clontech and Ariad.
A regulatable promoter comprising a repressor with an operator may be used. In one embodiment, the lac repressor from E.coli may act as a transcription regulator to regulate transcription of a mammalian Cell promoter harboring the lac operon (M.Brown et al, cell,49:603-612 (1987); gossen and Bujard (1992); M.Gossen et al, natl. Acad. Sci. USA,89:5547-5551 (1992)), and the tetracycline repressor (tetR) is combined with a transcriptional activator (VP 16) to produce a tetR-mammalian Cell transcriptional activator fusion protein tTa (tetR-VP 16), wherein the minimal promoter harboring tetO is derived from a human cytomegalovirus (hCMV) major immediate early promoter to produce the tetR-tet operon gene system, thereby controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operon is correctly located downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivative, may act as a potent trans regulator to regulate gene expression in mammalian cells (Yao et al, human Gene Therapy,10 (16): 1392-1399 (2003)). A particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivators or repressor fusion proteins (which in some cases may be cytotoxic) (Gossen et al, natl. Acad. Sci. USA,89:5547-5551 (1992); shock et al, proc. Natl. Acad. Sci. USA,92:6522-6526 (1995)) to achieve its regulatory effect.
In addition, the carrier may contain, for example, some or all of the following: selectable marker genes, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences for high level transcription from immediate early genes of human CMV; transcription termination and RNA processing signals for mRNA stability from SV 40; SV40 polyoma viral origin of replication and ColE1 for appropriate episomal replication; an internal ribosome binding site (IRESE); a multifunctional multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNAs. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.
Examples of polyadenylation signals that may be used to practice the methods described herein include, but are not limited to, human collagen I polyadenylation signals, human collagen II polyadenylation signals, and SV40 polyadenylation signals.
One or more vectors (e.g., expression vectors) comprising nucleic acids encoding any of the antibodies can be introduced into a suitable host cell to produce the antibodies. The host cell may be cultured under suitable conditions for expressing the antibody or any polypeptide chain thereof. Such antibodies or polypeptide chains thereof may be recovered from the cultured cells (e.g., from the cells or culture supernatant) via conventional methods (e.g., affinity purification). If desired, the polypeptide chains of the antibodies may be incubated under suitable conditions for a suitable period of time to allow for the production of the antibodies.
In some embodiments, the methods for making the antibodies described herein involve recombinant expression vectors encoding the heavy and light chains of an anti-galectin-9 antibody as also described herein. The recombinant expression vector may be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Positive transformant host cells can be selected and cultured under suitable conditions that allow expression of the two polypeptide chains forming the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cell may be incubated under suitable conditions that allow for antibody formation.
In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-galectin-9 antibody and the other encoding the light chain of an anti-galectin-9 antibody. Both recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods (e.g., calcium phosphate-mediated transfection). Alternatively, each of the expression vectors may be introduced into a suitable host cell. Positive transformants may be selected and cultured under suitable conditions that allow expression of the polypeptide chain of the antibody. When both expression vectors are introduced into the same host cell, the antibodies produced therein may be recovered from the host cell or medium. If desired, the polypeptide chain may be recovered from the host cell or culture medium and then cultured under suitable conditions that allow for antibody formation. When two expression vectors are introduced into different host cells, each of them may be recovered from the corresponding host cell or from the corresponding medium. The two polypeptide chains may then be incubated under suitable conditions for antibody formation.
Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture the host cells and recover the antibodies from the culture medium. For example, some antibodies can be isolated by affinity chromatography using protein a or protein G coupled matrices.
Any of a nucleic acid encoding the heavy chain, the light chain, or both of an anti-galectin-9 antibody as described herein, a vector (e.g., an expression vector) containing the nucleic acid, and a host cell comprising the vector is within the scope of the disclosure.
The anti-galectin-9 antibodies thus prepared may be characterized using methods known in the art, whereby a reduction, improvement or neutralization of galectin-9 biological activity is detected and/or measured. For example, in some embodiments, ELISA-type assays are suitable for qualitative or quantitative measurement of galectin-9 inhibition of Dectin-1 or TIM-3 signaling.
The biological activity of anti-galectin-9 antibodies can be verified by incubating the candidate antibodies with Dectin-1 and galectin-9 and monitoring any one or more of the following characteristics: (a) Binding between Dectin-1 and galectin-9 and inhibition of signal transduction mediated by said binding; (b) preventing, ameliorating or treating any aspect of a solid tumor; (c) blocking or reducing Dectin-1 activation; (d) Inhibit (reduce) synthesis, production or release of galectin-9. Alternatively, TIM-3 can be used to verify the biological activity of anti-galectin-9 antibodies using the protocol described above. Alternatively, CD206 may be used to verify the biological activity of anti-galectin-9 antibodies using the protocol described above.
In some embodiments, biological activity or efficacy in a subject is assessed, for example, by measuring peripheral and intratumoral T cell ratios, T cell activation, or by macrophage typing.
Other assays to determine the biological activity of anti-galectin-9 antibodies include measuring cd8+ and cd4+ (conventional) T cell activation (in vitro or in vivo assays, e.g., by measuring inflammatory cytokine levels such as ifnγ, tnfα, CD44, ICOS granzyme B, perforin, IL2 (up-regulation); CD26L and IL-10 (down-regulation)); measurement of macrophages re-programming (in vitro or in vivo) from, for example, the M2 to M1 phenotype (e.g., increased mhc ii, decreased CD206, increased TNF- α, and iNOS). Alternatively, the level of ADCC may be assessed in an in vitro assay as described herein.
Combination cancer therapy
The present disclosure provides methods of using any of the anti-galectin antibodies, e.g., G9.2-17 (IgG 4)), with one or more chemotherapeutic agents such as gemcitabine and/or paclitaxel (e.g.,) Methods of treating solid tumors, such as Pancreatic Ductal Adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal Cell Carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other GI solid tumors, in combination.
Without being bound by theory, it is believed that the anti-galectin-9 antibodies may reprogram immune responses against tumor cells by inhibiting Dectin-1, for example, via inhibiting activity of γδ T cells infiltrating into the tumor microenvironment and/or by enhancing immune surveillance against tumor cells, for example, by activating cd4+ and/or cd8+ T cells. Thus, it is expected that the combined use of an anti-galectin-9 antibody and one or more chemotherapeutic agents (such as those described herein) will significantly enhance anti-tumor efficacy.
(A) Exemplary target solid tumors for treatment
In some aspects, the present disclosure provides methods of treating solid tumors, such as PDAC, CRC, HCC, cholangiocarcinoma, renal Cell Carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or other GI solid tumors. The methods of treatment disclosed herein involve combination therapy of an anti-Gal 9 antibody such as G9.2-17 (IgG 4) and one or more chemotherapeutic agents (e.g., gemcitabine and paclitaxel as disclosed herein).
Pancreatic Ductal Adenocarcinoma (PDAC) is a devastating disease with few long-term survivors (Yadav et al, gastrology, 2013,144,1252-1261). Inflammation is extremely important in the progression of PDAC, as in the absence of concomitant inflammation, oncogenic mutations alone are insufficient to cause tumorigenesis (Guerra et al, cancer Cell,2007,11,291-302). Congenital and adaptive immunity synergistically promote tumor progression in PDACs. In particular, tumors Specific innate immune subpopulations within The Microenvironment (TME) are prone to culture adaptive immune effector cells into tumor-permissive phenotypes. Antigen Presenting Cell (APC) populations, including M2 polarized tumor-associated macrophages (TAM) and myeloid Dendritic Cells (DC), induce the production of immunosuppressive Th2 cells that favor tumor-protective Th1 cells (Ochi et al, J of Exp Med.,2012,209,1671-1687; zhu et al, cancer Res.,2014,74,5057-5069). Similarly, myeloid-derived suppressor cells (MDSCs) have been shown to eliminate anti-tumor CD8 in PDAC + Cytotoxic T Lymphocytes (CTL) respond and promote metastatic progression (Connolly et al, J Leuk biol.,2010,87,713-725; pylayeva-Gupta et al, cancer Cell,2012,21,836-847; bayne et al, cancer Cell,2012,21,822-835).
Colorectal cancer (CRC) (also known as bowel cancer, colon cancer or rectal cancer) is any cancer that affects the colon and rectum. CRC is known to be driven by genetic alterations of tumor cells and is also affected by tumor-host interactions. Recent reports have demonstrated a direct correlation between the density of certain T lymphocyte subpopulations and favorable clinical outcomes in CRC, supporting the major role of T cell mediated immunity in inhibiting tumor progression of CRC.
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Hepatocellular carcinoma most commonly occurs in people with chronic liver disease, such as cirrhosis caused by hepatitis b or hepatitis c infection. HCC is often associated with cirrhosis and extensive lymphocyte infiltration due to chronic viral infection. Many studies have demonstrated that tumor-infiltrating effector cd8+ T cells and T helper 17 (Th 17) cells are associated with increased survival after surgical removal of tumors. However, tumor infiltrating effector T cells are unable to control tumor growth and metastasis (Pang et al Cancer Immunol Immunother 2009; 58:877-886).
Bile duct cancer is a group of cancers that begin in the bile duct. Cholangiocarcinomas are typically classified according to their location relative to the liver. For example, intrahepatic cholangiocarcinoma accounts for less than 10% of all cholangiocarcinoma cases, beginning in the small bile duct within the liver. In another example, portal cholangiocarcinoma (also known as klotskin tumor) accounts for more than half of the cholangiocarcinoma cases, beginning in the portal where the two main bile ducts merge and leave the liver. Others are classified as distal bile duct cancers, beginning in the extrahepatic bile duct.
In some embodiments, the methods of the disclosure increase antitumor activity (e.g., decrease cell proliferation, tumor growth, tumor volume and/or tumor burden or burden, or decrease the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more compared to the level in the pre-treatment or control subject. In some embodiments, the decrease is measured by comparing the cell proliferation, tumor growth, and/or tumor volume of the subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating cancer in a subject allows for amelioration of one or more symptoms of the cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the cancer cells and/or biomarkers in the subject are measured in a biological sample such as blood, serum, plasma, urine, peritoneal fluid, and/or biopsies from tissues or organs before, during, and after administration of the pharmaceutical composition. In some embodiments, the methods comprise administering a composition of the invention to reduce the tumor volume, size, load, or burden in the subject to an undetectable size, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the tumor volume, size, load, or burden in the subject prior to treatment. In other embodiments, the methods comprise administering a composition of the invention to reduce the cell proliferation rate or tumor growth rate of the subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the pre-treatment rate. In other embodiments, the methods comprise administering a composition of the invention to reduce the progression of a metastatic focus or the number or size of metastatic lesions in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the rate prior to treatment.
The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within an acceptable standard deviation. Alternatively, "about" may mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, more preferably up to ±1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude of the value, preferably within 2 times the value. Where a particular value is described in the application and claims, unless otherwise indicated, the term "about" is implicit and, in this context, means within an acceptable error range for the particular value.
As used herein, the term "treating" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from, or prone to, a target disease or disorder, a symptom of a disease/disorder, with the aim of curing, healing, alleviating, moderating, altering, remediating, alleviating, ameliorating, or affecting the disorder, the symptom of a disease or disorder, or the propensity to a disease or disorder.
Alleviation of a target disease/disorder includes delaying the development or progression of the disease, or reducing the severity of the disease or extending survival. The alleviation of the disease or the prolongation of the survival does not necessarily require a cure. As used herein, "delay" of progression of a target disease or disorder refers to delaying, impeding, slowing, delaying, stabilizing, and/or slowing the progression of the disease. This delay may be of different lengths of time, depending on the history of the disease and/or the individual being treated. A method of "delaying" or alleviating the progression of a disease or delaying the onset of a disease is a method of reducing the likelihood of one or more symptoms of a disease occurring within a given time frame and/or alleviating the extent of symptoms within a given time frame, as compared to the absence of the method. Such comparisons are typically based on clinical studies using a number of subjects sufficient to yield statistically significant results.
"progression" or "progression" of a disease refers to the initial manifestation and/or subsequent progression of the disease. The progression of the disease can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progress that may not be detectable. For the purposes of this disclosure, development or progression refers to the biological process of symptoms. "progression" includes occurrence, recurrence and onset. As used herein, a "seizure" or "occurrence" of a disease or disorder of interest includes a first seizure and/or recurrence.
(B) Exemplary patient populations for treatment
Subjects with target solid tumors such as PDACs as disclosed herein can be identified by routine medical examinations (e.g., laboratory tests, organ function tests, genetic tests, interventional procedures (biopsies, surgery), any and all related imaging modalities). In some embodiments, the subject to be treated by the methods described herein is a human cancer patient who has undergone or is undergoing an anti-cancer therapy (e.g., chemotherapy, radiation therapy, immunotherapy, tumor treatment domain (TT domain) or surgery).
In some embodiments, the subject has received a prior immunomodulatory anti-tumor agent. Non-limiting examples of such immunomodulators include, but are not limited to, anti-PD 1, anti-PD-L1, anti-CTLA-4, anti-OX 40, anti-CD 137, anti-TIGIT, anti-PVRIG, platinum-based agents, and the like. Non-limiting examples of platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, and lobaplatin. In some embodiments, the subject shows disease progression by treatment. In other embodiments, the subject is resistant to treatment (either from the head or obtained). In some embodiments, such subjects are demonstrated to have advanced malignancy (e.g., inoperable or metastatic). Alternatively, or in addition, in some embodiments, the subject has no standard treatment options available or is not in compliance with the conditions of the standard treatment options, which refer to therapies commonly used in the clinical setting to treat the corresponding solid tumor.
Tumor therapeutic electric fields (TTFields) are a type of cancer treatment that uses alternating electric fields of medium frequency (-100-500 kHz) and low intensity (1-3V/cm) to disrupt cell division. In any of the embodiments described herein, the anti-galectin-9 antibody can be administered prior to, concurrently with, or after a tumor treatment domain (TT domain) regimen in combination with a chemotherapeutic agent described herein. In any of the embodiments described herein, the anti-galectin-9 antibody can be administered in combination with a chemotherapeutic agent described herein prior to, concurrently with, or after a therapy based on a reverse thermal hydrogel technique (e.g., reverse thermal hydrogel chemotherapy).
In some cases, the subject may be a human patient with a refractory disease (e.g., refractory PDAC). As used herein, "refractory" refers to a tumor that does not respond to treatment or becomes resistant. In some cases, the subject may be a human patient with a recurrent disease (e.g., recurrent PDAC). As used herein, "recurrent" or "recurrence" refers to a tumor that reappears or progresses after a period of treatment improvement (e.g., partial or complete response).
In some embodiments, a human patient to be treated by the methods disclosed herein may meet one or more of the inclusion and exclusion criteria disclosed in example 2 below. For example, a human patient may be older than 18 years and have a histologically confirmed unresectable metastatic cancer (e.g., adenocarcinoma and squamous cell carcinoma). The patient may have a disease that is measurable according to RECIST v.1.1. In some cases, a human patient may have a recently archived tumor sample (e.g., obtained within 5 years) that can be used for biomarker analysis (e.g., galectin-9 tumor tissue expression, which can be assessed by IHC). In some cases, the human patient is a PDAC patient that has received at least one line of systemic therapy in a metastatic cancer setting. Such patients may not have received a gemcitabine-containing regimen or have not been treated with a gemcitabine-containing regimen for at least 6 months. The patient may have an eastern tumor cooperative group (ECOG) performance status of 0-1 and/or Karnofsky score>70. The patient may also have sufficient hematologic and end organ function, e.g., neutrophil count ≡1X10 9 Platelet count 100x10 9 /LFor HCC in part 1, 50x10 9 L; hemoglobin not transfused for the previous week is not less than 9.0g/dL, creatinine is not more than 1.5xULN, AST (SGOT) is not more than 3xULN (not more than 5xULN when HCC or liver metastasis is present), ALT (SGPT) is not more than 3xULN (not more than 5xULN when HCC or liver metastasis is present), bilirubin is not more than 1.5xULN (bilirubin known to have not more than 3.0xULN for patients with Gilbert's disease), albumin is not less than 3.0g/dL, INR and PTT are not more than 1.5xULN; and/or amylase and lipase are less than or equal to 1.5xULN. In some cases, the human patient showed no evidence of active infection or infection requiring parenteral antibiotics, and no severe infection within 4 weeks prior to initiation of treatment. Pancreatic, biliary or intestinal fistulae are allowed, provided that they are controlled with an appropriate uninfected and unobstructed drainage tube.
Alternatively or additionally, a human patient undergoing any of the treatments disclosed herein may not have the following: (i) With primary, non-inventive metastatic cancer, (ii) with clinically significant, actively uncontrolled bleeding, any bleeding constitutions (e.g., active peptic ulcer); (iii) Radiation therapy within 4 weeks of treatment at the first dose, (iv) with a mushroom tumor mass or locally advanced PDAC; (v) Since previous cancer therapies have toxicity (except for alopecia and vitiligo) of ≡ctcae grade 3; (v) There is a second history of malignancy, (vi) evidence of serious or uncontrolled systemic disease, congestive heart failure, grade 2 of the New York Heart Association (NYHA), or Myocardial Infarction (MI) occurring within 6 months, (vii) severe non-healing wounds, active ulcers, or untreated fractures; (viii) Uncontrolled pleural effusion, pericardial effusion or ascites, requiring repeated drainage procedures; (ix) Has a history of severe allergic, anaphylactic or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins; (x) Significant vascular disease (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis) within 6 months after treatment, a history of pulmonary embolism, stroke or transient ischemic attacks within the first 3 months of treatment, and/or a history of abdominal fistulae or gastrointestinal perforation within 6 months of treatment; (xi) With active autoimmune disorders (type I diabetes, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis or alopecia excluded); (xii) in need of systemic immunosuppressive therapy; (xii) Tumor-associated pain (> grade 3), unresponsive to extensive analgesic interventions (oral and/or patches); (xiii) Uncontrolled hypercalcemia despite the use of bisphosphonates; (xiv) receiving an organ transplant.
In some cases, the subject is a human patient having elevated levels of galectin-9 as compared to control levels. The level of galectin-9 may be a plasma or serum level of galectin-9 in a human patient. In other examples, the level of galectin-9 may be a level of galectin-9 on a cell surface, e.g., a level of galectin-9 on a cancer cell. In one example, the level of galectin-9 may be a level of surface galectin-9 expressed on cancer cells in patient-Derived Organotypic Tumor Spheroids (PDOTs), which may be prepared, for example, by the methods disclosed in the examples below. Control levels may refer to the level of galectin-9 in a matched sample of a subject of the same species (e.g., human) without a solid tumor. In some examples, the control level represents a level of galectin-9 in a healthy subject.
To identify such subjects, a suitable biological sample may be obtained from a subject suspected of having a solid tumor, and the biological sample may be analyzed to determine the level of galectin-9 (e.g., free, cell surface expressed or all) contained therein using conventional methods such as ELISA or FACS. In some embodiments, for example, organoid cultures are prepared as described herein and used to assess galectin-9 levels in a subject. Single cells obtained from certain fractions obtained as part of the organoid preparation process are also useful for assessing galectin-9 levels in a subject. In some cases, the assay for measuring the level of galectin-9 in free form or expressed on the cell surface involves the use of antibodies that specifically bind to galectin-9 (e.g., specifically bind to human galectin-9). Any anti-galectin-9 antibody known in the art can be tested for suitability in any of the assays described above and then used in such assays in a conventional manner. In some embodiments, the antibodies described herein (e.g., G9.2-17 antibodies) can be used, such as in an assay. In some embodiments, antibodies are described in co-pending U.S. patent application 16/173,970 and co-owned co-pending international patent application PCT/US18/58028, the respective relevant disclosures of each of which are incorporated by reference for the purposes and subject matter cited herein. In some examples, the anti-galectin-9 antibody is a Fab molecule. Assays for determining galectin-9 levels as disclosed herein are also within the scope of the present disclosure.
(C) Exemplary treatment conditions
In some embodiments, an antibody described herein (e.g., G9.2-17, such as an IgG4 form thereof) is administered to a subject in need of treatment in an amount sufficient to inhibit galectin-9 (and/or Dectin-1 or TIM-3 or CD 206) by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo in an immunosuppressive immune cell in a tumor. In other embodiments, an antibody described herein (e.g., G9.2-17) is administered in an amount effective to reduce the level of activity of galectin-9 (and/or Dectin-1 or TIM-3 or CD 206) in immunosuppressive immune cells in a tumor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) compared to the level in a pre-treatment or control subject. In some embodiments, an antibody described herein (e.g., G9.2-17) is administered to a subject in need of treatment in an amount sufficient to promote M1-like programming in TAMs in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) compared to the level in the pre-treatment or control subject.
Depending on the type of disease or the site of the disease to be treated, the pharmaceutical composition may be administered to the subject using conventional methods known to those of ordinary skill in the medical arts. In some embodiments, the anti-galectin-9 antibody may be administered to the subject by intravenous infusion.
The injectable composition may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol and polyols (glycerol, propylene glycol, liquid polyethylene glycols, etc.). For intravenous infusion, the water-soluble antibody may be administered by instillation, thereby infusing a pharmaceutical formulation containing the antibody and physiologically acceptable excipients. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. Intramuscular formulations of antibodies, e.g., sterile formulations in the form of suitable soluble salts, may be dissolved in a pharmaceutical excipient (such as water for injection, 0.9% saline or 5% dextrose solution) and administered.
In some embodiments, methods are provided in which an anti-galectin-9 antibody is administered concurrently with one or more chemotherapeutic agents. In some embodiments, the anti-galectin-9 antibody is administered before or after one or more chemotherapeutic agents. In some embodiments, the one or more chemotherapeutic agents are administered systemically. In some embodiments, the one or more chemotherapeutic agents are administered topically. In some embodiments, the one or more chemotherapeutic agents are administered by intravenous administration (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intraarterial, intraarticular, intravesical, intrasynovial, intrathecal, intratumoral, subcutaneous, oral, inhalation, or topical routes). In one embodiment, the subject is administered one or more chemotherapeutic agents by intravenous infusion. In some embodiments, an anti-galectin-9 antibody described herein is administered to a patient who is currently receiving or has previously received an anti-cancer therapy (e.g., chemotherapy).
An effective amount of a pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment, either systemically or locally via a suitable route. In some embodiments, the anti-galectin-9 antibody is administered by intravenous administration (e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intraarterial, intraarticular, intrasynovial, intrathecal, intravesical, intratumoral, subcutaneous, oral, inhalation, or topical route). In one embodiment, the anti-galectin-9 antibody is administered to the subject by intravenous infusion. In one embodiment, the anti-galectin-9 antibody is administered intraperitoneally to a subject. As used herein, "effective amount" refers to the amount of each active agent required to impart a therapeutic effect to a subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is reduced galectin-9 activity and/or amount/expression, reduced Dectin-1 signaling, reduced TIM-3 signaling, reduced CD206 signaling, or an increased anti-tumor immune response in a tumor microenvironment. Non-limiting examples of increased anti-tumor responses include increased activation levels of effector T cells, or a transition of TAM from the M2 phenotype to the M1 phenotype. In some cases, the anti-tumor response comprises an increased ADCC response. It will be apparent to those skilled in the art that determining whether an amount of antibody achieves a therapeutic effect. As will be appreciated by those of skill in the art, the effective amount will vary depending upon the particular condition being treated, the severity of the condition, the individual patient parameters (including age, physical condition, body type, sex, and weight), the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with only routine experimentation. It is generally preferred to use the maximum dose of the individual components or combinations thereof, i.e. the highest safe dose according to sound medical judgment.
Empirical considerations (such as half-life) often help determine the dosage. For example, antibodies that are compatible with the human immune system (such as humanized antibodies or fully human antibodies) are used in some cases to extend the half-life of the antibody and to protect the antibody from attack by the host immune system. The frequency of administration may be determined and adjusted during the course of treatment and is generally, but not necessarily, based on the treatment and/or inhibition and/or amelioration and/or delay of the target disease/disorder. Alternatively, a sustained continuous release formulation of the antibody may be suitable. Various formulations and devices for achieving sustained release are known in the art.
In one example, the dosage of an antibody as described herein is empirically determined in an individual who has been administered one or more antibody administrations. The subject is administered increasing doses of the antagonist. To assess the efficacy of an antagonist, an index of disease/condition may be followed.
Anti-galectin-9 antibody treatment
Any of the anti-galectin-9 antibodies described herein can be used in any of the methods described herein. In some embodiments, the anti-galectin-9 antibody is G9.2-17. The G9.2-17 antibody may be an IgG4 molecule (G9.2-17 (IgG 4)) as disclosed herein. In a specific example, an anti-galectin-9 antibody (G9.2-17) as used herein has a heavy chain of SEQ ID NO. 19 and a light chain of SEQ ID NO. 15. The anti-Gal 9 antibodies can be formulated as disclosed herein and administered to a subject in need of treatment by a suitable route, such as intravenous infusion.
In some cases, an anti-galectin-9 antibody (e.g., G9.2-17, such as G9.2-17 (IgG 4)) as disclosed herein can be administered to a subject at a suitable dose (e.g., about 0.2 to about 32 mg/kg). Examples include 0.2mg/kg to 0.5mg/kg, 0.5mg/kg to 1mg/kg, 1mg/kg to 2mg/kg, 2mg/kg to 3mg/kg, 3mg/kg to 4mg/kg, 4mg/kg to 6mg/kg, 4mg/kg to 6.3mg/kg, 6mg/kg to 8mg/kg, 6.3mg/kg to 8mg/kg, 4mg/kg to 8mg/kg, 8mg/kg to 12mg/kg, 8mg/kg to 10mg/kg, 10mg/kg to 12mg/kg to 16mg/kg, 16mg/kg to 20mg/kg, 20mg/kg to 24mg/kg, 24mg/kg to 28mg/kg, or 28mg/kg to 32mg/kg (e.g., 0.2mg/kg, 0.5mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 6.3mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg or 32 mg/kg) or any of these. In some embodiments, the amount of the active agent is about 0.2mg/kg to 0.5mg/kg, 0.5mg/kg to 1mg/kg, about 1mg/kg to 2mg/kg, about 2mg/kg to 4mg/kg, about 4mg/kg to 8mg/kg, 4mg/kg to 6mg/kg, 4mg/kg to 6.3mg/kg, 6mg/kg to 8mg/kg, 6.3mg/kg to 8mg/kg, about 8mg/kg to 12mg/kg, about 12mg/kg to 16mg/kg, about 16mg/kg to 20mg/kg, about 20mg/kg to 24mg/kg, about 24mg/kg to 28mg/kg, or about 28mg/kg to 32mg/kg (e.g., an incremental dose of any of these or an incremental dose of any of these ranges of antibodies of about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg or about 32 mg/kg.
In some embodiments, an anti-Gal-9 antibody such as G9.2-17 (IgG 4) is administered at 0.2 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 0.6 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 0.63 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 2 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 4 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 6 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 6.3 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 8 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 10 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 12 mg/kg. In some embodiments, the anti-Gal-9 antibody is administered at 16 mg/kg. In some cases, multiple doses of anti-galectin-9 antibody may be administered to a subject at suitable intervals or periods (e.g., once a week, once every two to four weeks (e.g., every two, three, or four weeks)). The treatment may last for a suitable period of time, for example, up to 3 months, up to 6 months, or up to 12 months, or up to 24 months or longer. In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, an anti-Gal-9 antibody is administered to a subject at a dose of 10mg/kg once a week. Alternatively, the anti-Gal-9 antibody is administered to the subject at a dose of 16mg/kg once a week.
In some cases, an anti-Gal-9 antibody disclosed herein, such as G9.2-17 (IgG 4), may be administered to a subject at a steady dose (e.g., about 650mg to about 1120 mg) once per week to once every 4 weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at about 650mg to about 700mg once a week. In some examples, the anti-Gal-9 antibody is administered to the subject from about 650mg to about 700mg once every two weeks. In some examples, the anti-Gal-9 antibody is administered to the subject at about 1040mg to about 1120mg once a week. In some examples, the anti-Gal-9 antibody is administered to the subject from about 1040mg to about 1120mg once every two weeks.
In some examples, an anti-Gal-9 antibody such as a G9.2-17 (IgG 4) antibody is administered via intravenous infusion once every two weeks at a dose of about 3mg/kg to a human patient suffering from a solid tumor (e.g., PDAC) as disclosed herein. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 15mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 0.2mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 0.6mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 0.63mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 2mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 4mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 6mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 6.3mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 8mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 10mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor at a dose of about 12mg/kg via intravenous infusion once every two weeks. In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor via intravenous infusion at a dose of about 16mg/kg or higher at a dose level once every two weeks.
In other examples, the anti-galectin-9 antibody is administered to a human patient having a target solid tumor via intravenous infusion at a dose selected from the group consisting of 0.2mg/kg, 0.63mg/kg, 2mg/kg, 4mg/kg, 6mg/kg, 6.3mg/kg, 8mg/kg, 10mg/kg, 12mg/kg, and 16mg/kg or higher at a dose level once every two weeks. In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some examples, about 2mg/kg to 16mg/kg of the anti-Gal 9 antibody (e.g., G9.2-17 in the form of IgG 4) can be administered once every two weeks to a subject in need of treatment. In some examples, about 0.2mg/kg to 16mg/kg of the anti-Gal 9 antibody (e.g., G9.2-17 in the form of IgG 4) can be administered once every two weeks to a subject in need of treatment. In some examples, the anti-Gal 9 antibody (e.g., G9.2-17 in the form of IgG4 as disclosed herein, having the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO: 15) is administered to the subject at a dose of about 0.5mg/kg, 0.6mg/kg, 0.63mg/kg, 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg or any increment thereof) by intravenous infusion once a week. In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some examples, an anti-Gal 9 antibody (e.g., G9.2-17 in the form of IgG4 as disclosed herein, having the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO: 15) is administered to a subject at a dose of about 0.2mg/kg once every two weeks by intravenous infusion. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 0.6mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 0.63mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 2mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 4mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 6mg/kg once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 6.3mg/kg once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 8mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 10mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 12mg/kg by intravenous infusion once every two weeks. In some examples, the anti-Gal 9 antibody is administered to the subject at a dose of about 16mg/kg by intravenous infusion once every two weeks. In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some examples, the intravenous infusion is administered at 0.2mg/kg to 0.5mg/kg, 0.5mg/kg to 1mg/kg, about 1mg/kg to 2mg/kg, about 3mg/kg to 4mg/kg, about 4mg/kg to 6mg/kg, about 4mg/kg to 6.3mg/kg, about 4mg/kg to 8mg/kg, about 8mg/kg to 10mg/kg, about 8mg/kg to 12mg/kg, about 10mg/kg to 12mg/kg, about 12mg/kg to 16mg/kg, about 16mg/kg to 20mg/kg, about 20mg/kg to 24mg/kg, about 24mg/kg to 28mg/kg, or about 28mg/kg to 32mg/kg once per week (e.g., about 0.2mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg or about 32mg/kg or any of antibodies therein as disclosed herein or in any of the forms 9G (e.g) of anti-IgG 9 therein, having a heavy chain of SEQ ID NO. 19 and a light chain of SEQ ID NO. 15). In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some examples, the antibodies are administered in an amount of 0.2 to 0.5, 0.5 to 1, 1 to 2, 3 to 4, about 4 to 6, about 4 to 6.3, 4 to 8, 8 to 12, 8 to 10, 10 to 12, 12 to 16, 16 to 20, 20 to 24, 24 to 28, or 28mg to 32mg/kg (e.g., 0.2, 0.6, 0.63, 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 17, 25, 24, 22, 15, 19, 25, 26, 25, 15, 19, 15, or any of the incremental dosages of the subject in any of these ranges by intravenous infusion once a week, having a heavy chain of SEQ ID NO. 19 and a light chain of SEQ ID NO. 15). In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some examples, the anti-Gal 9 antibody (e.g., G9.2-17 in the form of IgG4 as disclosed herein, having the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO: 15) is administered to the subject at a dose of about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg, or any increment therein by intravenous infusion once a week. In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In specific examples, the anti-galectin 9 antibody, such as G9.2-17 (IgG 4), may be administered to a human patient at an appropriate dose (e.g., a dose disclosed herein) once a week. For example, 2.0mg/kg of G9.2-17 (IgG 4) may be administered to a human patient once a week. For example, 6.3mg/kg of G9.2-17 (IgG 4) may be administered once a week to a human patient. In another example, 10mg/kg of G9.2-17 (IgG 4) may be administered to a human patient once a week. Alternatively, 12mg/kg of G9.2-17 (IgG 4) may be administered to a human patient once a week. In yet another example, 16mg/kg of G9.2-17 (IgG 4) may be administered once a week to a human patient.
In some cases, the anti-galectin-9 antibody may be administered to a human patient for at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, or more. In some cases, the treatment period may be from 6 months to 12 months. In other cases, the treatment period may be 12 months to 24 months. In other cases, the treatment period may exceed 24 months.
In some embodiments, an anti-galectin 9 antibody disclosed herein (e.g., G9.2-17IgG 4) is administered intravenously over an infusion period of 30 minutes to 6 hours. In some examples, intravenous infusion of anti-galectin-9 antibody may be performed for 30 minutes to 2 hours. In other examples, the anti-galectin 9 antibody may be administered via a long infusion period (e.g., about 2-6 hours, e.g., about 2-4 hours or about 4-6 hours). In specific examples, exemplary anti-galectin-9 antibodies may be infused intravenously over a period of about 3 hours, about 4 hours, about 5 hours, or about 6 hours.
In a specific embodiment, the interval or period is 1 week. In a specific embodiment, the interval or period is 2 weeks. In some embodiments, the regimen is one cycle every 2 weeks, two cycles every 2 weeks, three cycles every 2 weeks, four cycles every 2 weeks, or more than four cycles every 2 weeks. In some embodiments, the treatment is for 1 to 3 months once every 2 weeks, 3 to 6 months once every 2 weeks, 6 to 12 months once every 2 weeks, or 12 to 24 months or more once every 2 weeks.
In a specific embodiment, the interval or period is 3 weeks. In some embodiments, the regimen is one cycle every 3 weeks, two cycles every 3 weeks, three cycles every 3 weeks, four cycles every 3 weeks, or more than four cycles every 3 weeks. In some embodiments, the treatment is for 1 to 3 months once every 3 weeks, 3 to 6 months once every 3 weeks, 6 to 12 months once every 3 weeks, or 12 to 24 months or more once every 3 weeks.
In particular embodiments, the interval or period is 4 weeks or more. In some embodiments, the regimen is one cycle every 4 weeks or more, two cycles every 4 weeks or more, three cycles every 4 weeks or more, four cycles every 4 weeks or more, or more than four cycles every 4 or more. In some embodiments, the treatment is for 1 to 3 months once every 4 weeks or more, for 3 to 6 months once every 4 weeks or more, for 6 to 12 months once every 4 weeks or more, or for 12 to 24 months or more once every 4 weeks or more. In some embodiments, the treatment is a combination of treatments at different times, e.g., a combination of 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks. In some embodiments, the treatment interval is adjusted according to the patient's response to the treatment. In some embodiments, the dose is adjusted according to the patient's response to treatment. In some embodiments, the dose varies between treatment intervals. In some embodiments, treatment may be temporarily stopped. In some embodiments, anti-galectin-9 therapy is temporarily discontinued. In some embodiments, chemotherapy is temporarily stopped. In some implementations, both are temporarily stopped. In any of these embodiments, the anti-Gal 9 antibody can be G9.2-17 in the form of IgG4 as disclosed herein, having the heavy chain of SEQ ID NO:19 and the light chain of SEQ ID NO: 15.
Alternatively, a human patient may begin with a low dose (e.g., 0.2mg/kg, 0.63mg/kg, or 2 mg/kg) of an anti-galectin-9 antibody disclosed herein, such as G9.2-17 (IgG 4). When no adverse effect is observed, the dosage of the antibody can be increased to, for example, 6.3mg/kg, 10mg/kg or 16mg/kg.
Chemotherapy treatment
One or more chemotherapeutic agents mayTo include antimetabolites, microtubule (e.g., tubulin) inhibitors, platinum agents, or combinations thereof. Antimetabolites include, for example, folic acid antagonists (e.g., methotrexate) and nucleotide analogs such as pyrimidine antagonists (e.g., 5-fluorouracil, floxuridine, cytarabine, capecitabine, and gemcitabine), purine antagonists (e.g., 6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (e.g., cladribine, fludarabine, and jetstretin). Microtubule Inhibitors (MIT) include for example paclitaxel (e.g.,) Docetaxel, vinblastine, vincristine, and vinorelbine.
In some examples, the antimetabolite used in the methods disclosed herein is gemcitabine, which may be administered by intravenous infusion. The amount of gemcitabine to be administered to a subject depends on many factors, including height and weight, general health or other health issues, and the type of cancer to be treated, which will be within the knowledge of the practitioner following the guidelines provided by the U.S. food and drug administration (see, for example, the drug label of an approved gemcitabine product). In some examples, 1000mg/m may be administered by intravenous infusion 2 Gemcitabine is administered to the subject, optionally once a week for up to 7 weeks within 30 minutes, followed by a week of rest from treatment. The subsequent cycle may include weekly infusions every three consecutive weeks every four weeks. If one or more adverse effects occur, the dose of gemcitabine may be reduced or the treatment may be discontinued. Further details regarding managing adverse effects associated with gemcitabine treatment are provided in example 2 below.
Microtubule inhibitors are a class of compounds that inhibit cell microtubule formation, thereby blocking cell proliferation. In some examples, the microtubule inhibitor is a stabilizer that promotes microtubule polymerization. Examples include taxanes and epothilones. In other examples, the microtubule inhibitor is a destabilizing agent that promotes microtubule depolymerization. Examples include vinca alkaloids. In some examples, the microtubule inhibitor used in the methods disclosed herein is paclitaxel. At the position ofIn some cases, paclitaxel is in free form. In other cases, paclitaxel is conjugated to a protein such as albumin. In a specific example, the paclitaxel isWhich is nanoparticulate albumin conjugated paclitaxel.
The amount of paclitaxel (e.g., protein-bound paclitaxel, such as nab-paclitaxel) to be administered to a subject depends on many factors, including height and weight, general health or other health issues, and the type of cancer to be treated, which will be within the knowledge of the practitioner following the guidelines provided by the U.S. food and drug administration (e.g., see the drug label of an approved paclitaxel product). For example, when nanoparticle albumin conjugated paclitaxel (nab-paclitaxel, for example, ) At this time, 260mg/m may be infused intravenously every 3 weeks over 30 minutes 2 The subject is administered. If serious adverse effects (e.g., neutropenia or severe sensory neuropathy) are observed, the dose of paclitaxel may be reduced. In some cases, the dosage of nab-paclitaxel may be reduced to 180mg/m 2 . When combined with anti-Gal 9 antibody, the dose of paclitaxel may be 125mg/m 2 . If desired, the paclitaxel dose can be reduced to 100mg/m 2 Or 75mg/m 2 . Further details regarding management of paclitaxel-related side effects are provided in example 2 below.
In some cases, the chemotherapeutic agent used with the anti-Gal-9 antibody may include a platinum agent, such as cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatinum tetranitrate, phenanthreneplatinum, picoplatin, or satraplatin.
Combination therapy
Combination therapies provided herein include any anti-galectin-9 antibody therapy disclosed herein (e.g., antibodies directed to G9.2-17 (IgG 4)) and any chemotherapy disclosed herein (e.g., directed to a combination of gemcitabine and paclitaxel).
In some embodiments, the anti-Gal 9 antibody (e.g., G9.2-17 in the IgG4 format), gemcitabine, and paclitaxel (e.g., nanoparticulate albumin conjugated paclitaxel or ) The treatment regimen and dosing regimen provided in example 2 below may be administered to a subject in need of treatment. For example, the treatment may include one or more cycles, each cycle consisting of 28 days. In each cycle, an anti-Gal 9 antibody (e.g., G9.2-17 (IgG 4)) is administered to a subject (e.g., a human patient with PDAC) via intravenous infusion at a dose of about 2mg/kg to 16mg/kg (e.g., about 2mg/kg, about 4mg/kg, about 8mg/kg, about 12mg/kg, or about 16 mg/kg) once every two weeks (e.g., on days 1 and 15). In some embodiments, the anti-Gal 9 antibody (e.g., G9.2-17 (IgG 4)) is administered to the subject (e.g., a human patient with PDAC) via intravenous infusion at a dose of about 0.2mg/kg to 16mg/kg (e.g., about 0.2mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 2mg/kg, about 4mg/kg, about 6mg/kg, about 6.3mg/kg, about 10mg/kg, or about 16 mg/kg) once every two weeks (e.g., on days 1 and 15). Gemcitabine and paclitaxel (e.g., protein-bound paclitaxel, such as ∈x), may be administered to a subject once a week for three weeks, and then one week without treatment (e.g., on days 1, 8, and 15 of a 28 day cycle) using dosages and dosing regimens as approved by the FDA >). For example, 1000mg/m may be infused via intravenous infusion once a week in each cycle 2 Gemcitabine is administered to a subject and may be administered at 125mg/m once a week 2 Paclitaxel is administered to the subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel may be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the uses described hereinMethods for treating a solid tumor (e.g., PDAC) include one or more treatment cycles of 28 days, wherein the anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 0.2mg/kg to about 32mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)), and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, at 125mg/m 2 Paclitaxel is administered intravenously (e.g., intravenous infusion) to the subject. In some examples, at 1000mg/m 2 Gemcitabine is administered (e.g., intravenously infused) intravenously to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel may be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein
(1) About 0.2mg/kg to 0.5mg/kg, about 0.5mg/kg to 1mg/kg, about 1mg/kg to 2mg/kg, about 3mg/kg to 4mg/kg, about 4mg/kg to 8mg/kg, about 4mg/kg to 6mg/kg, about 4mg/kg to 6.3mg/kg, about 6mg/kg to 8mg/kg, about 6.3mg/kg to 8mg/kg, about 8mg/kg to 12mg/kg, about 8mg/kg to 10mg/kg, about 10mg/kg to 12mg/kg, about 12mg/kg to 16mg/kg, about 16mg/kg to 20mg/kg, about 20mg/kg to 24mg/kg, about 24mg/kg to 28mg/kg, or about 28mg/kg to 32mg/kg via intravenous infusion on days 1 and 15 (i.e.2 weeks), about 0.2mg/kg, about 0.5mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about, about 31mg/kg or about 32 mg/kg) or any increment thereof,
(2) 1000mg/m on days 1, 8 and 15 2 Is subjected to the dosage direction of (2)The subject administers (e.g., intravenously injects) gemcitabine intravenously. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 ，
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject. When needed, the dosage of paclitaxel can be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein
(1) From 0.2mg/kg to 0.5mg/kg, from 0.5mg/kg to 1mg/kg, from 1mg/kg to 2mg/kg, from 3mg/kg to 4mg/kg, from 4mg/kg to 8mg/kg, from 4mg/kg to 6mg/kg, from 4mg/kg to 6.3mg/kg, from 6mg/kg to 8mg/kg, from 6.3mg/kg to 8mg/kg, from 8mg/kg to 12mg/kg, from 8mg/kg to 10mg/kg, from 10mg/kg to 12mg/kg, from 12mg/kg to 16mg/kg, from 16mg/kg to 20mg/kg, from 20mg/kg to 24mg/kg, from 24mg/kg to 28mg/kg, or from 28mg/kg to 32mg/kg via intravenous infusion on days 1 and 15 (i.e.2 w), A dose of 0.2mg/kg, 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 6.3mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg or 32 mg/kg) or any increment thereof is administered to a subject,
(2) 1000mg/m on days 1, 8 and 15 2 Gemcitabine is administered intravenously (e.g., by intravenous injection) to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 ，
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject. When needed, the dosage of paclitaxel can be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the methods described herein for treating a solid tumor (e.g., PDAC) comprise one or more treatment cycles of 28 days, wherein an anti-Gal 9 antibody is administered to a subject via intravenous infusion at a dose of about 2mg/kg to about 16mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)), and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some embodiments, the methods described herein for treating a solid tumor (e.g., PDAC) comprise one or more treatment cycles of 28 days, wherein the anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 0.2mg/kg to about 16mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)), and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15. In some examples, at 125mg/m 2 Paclitaxel is administered intravenously (e.g., intravenous infusion) to the subject. In some examples, at 1000mg/m 2 Gemcitabine is administered (e.g., intravenously infused) intravenously to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel may be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the methods described herein for treating a solid tumor (e.g., PDAC) comprise one or more treatment cycles of 28 days, wherein the anti-antibody is administered to the subject via intravenous infusion at a dose of about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg or about 20mg/kg, or any of these via intravenous infusion on days 1 and 15 days, and the combination of paclitaxel to the subject on days, e.g., 15, and the nanoparticles on days of the day 1 and 15. In some examples, at 125 mg/m 2 Paclitaxel is administered intravenously (e.g., intravenous infusion) to the subject. In some examples, at 1000mg/m 2 Gemcitabine is administered (e.g., intravenously infused) intravenously to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) may be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein
(1) About 0.2mg/kg to 0.5mg/kg, about 0.5mg/kg to 1mg/kg, about 1mg/kg to 2mg/kg, about 3mg/kg to 4mg/kg, about 4mg/kg to 8mg/kg, about 4mg/kg to 6mg/kg, about 4mg/kg to 6.3mg/kg, about 6mg/kg to 8mg/kg, about 6.3mg/kg to 8mg/kg, about 8mg/kg to 12mg/kg, about 8mg/kg to 10mg/kg, about 10mg/kg to 12mg/kg, about 12mg/kg to 16mg/kg, about 16mg/kg to 20mg/kg, about 20mg/kg to 24mg/kg, about 24mg/kg to 28mg/kg, or about 28mg/kg to 32mg/kg via intravenous infusion on days 1 and 15 (i.e.2 weeks), about 0.2mg/kg, about 0.5mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about, about 31mg/kg or about 32 mg/kg) or any increment thereof,
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) From 0.2mg/kg to 0.5mg/kg, from 0.5mg/kg to 1mg/kg, from 1mg/kg to 2mg/kg, from 3mg/kg to 4mg/kg, from 4mg/kg to 8mg/kg, from 4mg/kg to 6mg/kg, from 4mg/kg to 6.3mg/kg, from 6mg/kg to 8mg/kg, from 6.3mg/kg to 8mg/kg, from 8mg/kg to 12mg/kg, from 8mg/kg to 10mg/kg, from 10mg/kg to 12mg/kg, from 12mg/kg to 16mg/kg, from 16mg/kg to 20mg/kg, from 20mg/kg to 24mg/kg, from 24mg/kg to 28mg/kg, or from 28mg/kg to 32mg/kg via intravenous infusion on days 1 and 15 (i.e.2 w), A dose of 0.2mg/kg, 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 6.3mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg or 32 mg/kg) or any increment thereof is administered to a subject,
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein
(1) An anti-antibody dose of about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 31mg/kg, about 32mg/kg, or any of the subjects is administered via intravenous infusion on days 1 and 15 (i.e., once every 2 weeks),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) Administering an anti-Gal 9 antibody to the subject via intravenous infusion at a dose of about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg or any increment therein on days 1 and 15 (i.e., once every 2 weeks),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 0.2mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously to the subject (e.g.,intravenous infusion) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel).
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 0.6mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 0.63mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 2mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Intravenous administration (e.g., intravenous infusion) of paclitaxel to a subject(e.g., nanoparticle albumin-bound paclitaxel).
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 4mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 6mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 6.3mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Intravenous administration (e.g., intravenous infusion) of paclitaxel (e.g., nanoparticle albumin) to a subjectWhite bound paclitaxel).
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 8mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 10mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day period treatments, wherein:
(1) anti-Gal 9 antibody is administered to the subject via intravenous infusion at a dose of about 12mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 16mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) anti-Gal 9 antibody was administered to subjects via intravenous infusion at a dose of about 32mg/kg on days 1 and 15 (i.e., once every 2 weeks (q 2 w)),
(2) 1000mg/m on days 1, 8 and 15 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 125mg/m at day 1, day 8 and day 15 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In any of the above administration method embodiments, the dosage of gemcitabine may be reduced to 800mg/m when desired 2 Or 600mg/m 2 And alternatively or additionally, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) may be reduced to 100mg/m 2 Or 75mg/m 2 。
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) About 0.2mg/kg to 0.5mg/kg, about 0.5mg/kg to 1mg/kg, about 1mg/kg to 2mg/kg, about 3mg/kg to 4mg/kg, about 4mg/kg to 8mg/kg, about 4mg/kg to 6mg/kg, about 4mg/kg to 6.3mg/kg, about 6mg/kg to 8mg/kg, about 6.3mg/kg to 8mg/kg, about 8mg/kg to 12mg/kg, about 8mg/kg to 10mg/kg, about 10mg/kg to 12mg/kg, about 12mg/kg to 16mg/kg, about 16mg/kg to 20mg/kg, about 20mg/kg to 24mg/kg, about 24mg/kg to 28mg/kg, or about 28mg/kg to 32mg/kg via intravenous infusion on days 1 and 15 (i.e.2 weeks), about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about, about 31mg/kg or about 32 mg/kg) or any increment thereof,
(2) 800mg/m at day 1, day 8 and day 15 2 、600mg/m 2 Or 1000mg/m 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 100mg/m at day 1, day 8 and day 15 2 、75mg/m 2 Or 125mg/m 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) From 0.2mg/kg to 0.5mg/kg, from 0.5mg/kg to 1mg/kg, from 1mg/kg to 2mg/kg, from 3mg/kg to 4mg/kg, from 4mg/kg to 8mg/kg, from 4mg/kg to 6mg/kg, from 4mg/kg to 6.3mg/kg, from 6mg/kg to 8mg/kg, from 6.3mg/kg to 8mg/kg, from 8mg/kg to 12mg/kg, from 8mg/kg to 10mg/kg, from 10mg/kg to 12mg/kg, from 12mg/kg to 16mg/kg, from 16mg/kg to 20mg/kg, from 20mg/kg to 24mg/kg, from 24mg/kg to 28mg/kg, or from 28mg/kg to 32mg/kg via intravenous infusion on days 1 and 15 (i.e.2 w), A dose of 0.2mg/kg, 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 6.3mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg or 32 mg/kg) or any increment or therein is administered to a subject,
(2) 800mg/m at day 1, day 8 and day 15 2 、600mg/m 2 Or 1000mg/m 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 100mg/m at day 1, day 8 and day 15 2 、75mg/m 2 Or 125mg/m 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In some embodiments, the method comprises one or more 28-day treatment cycles, wherein:
(1) Administering an anti-Gal 9 antibody to the subject via intravenous infusion at a dose of about 0.2mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg, or any increment thereof, on days 1 and 15 (i.e., once every 2 weeks (q 2 w)).
(2) 800mg/m at day 1, day 8 and day 15 2 、600mg/m 2 Or 1000mg/m 2 Is administered intravenously (e.g., intravenous infusion) of gemcitabine to a subject,
(3) 100mg/m at day 1, day 8 and day 15 2 、75mg/m 2 Or 125mg/m 2 Is administered intravenously (e.g., infused intravenously) paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to a subject.
In any of the above methods of administration, the treatment period may last for a period of 12-24 months.
In any of the method embodiments described herein, the anti-galectin 9 antibody (alone or in combination with one or more chemotherapeutic agents such as gemcitabine and nab-paclitaxel, e.g., at the dosages described herein) may be administered once every 2 weeks for one cycle, once every 2 weeks for two cycles, once every 2 weeks for three cycles, once every 2 weeks for four cycles, or once every 2 weeks for more than four cycles. In some embodiments, the treatment is 1 to 3 months, 3 to 6 months, 6 to 12 months, 12 to 24 months, or longer. In some embodiments, the treatment is for 1 to 3 months once every 2 weeks, 3 to 6 months once every 2 weeks, 6 to 12 months once every 2 weeks, or 12 to 24 months or more once every 2 weeks.
In some embodiments, the methods described herein for treating a solid tumor (e.g., PDAC) comprise one or more treatment cycles of 28 days, wherein about 0.2mg/kg to 0.5mg/kg, about 0.5mg/kg to 1mg/kg, about 1mg/kg to 2mg/kg, about 3mg/kg to 4mg/kg, about 4mg/kg to 8mg/kg, about 4mg/kg to 6mg/kg, about 4mg/kg to 6.3mg/kg, about 6mg/kg to 8mg/kg, about 6.3mg/kg to 8mg/kg, about 8mg/kg to 12mg/kg, about 8mg/kg to 10mg/kg, about 10mg/kg to 12mg/kg, about 12mg/kg to 16mg/kg, about 16mg/kg to 20mg/kg, about 24mg/kg to 24mg/kg, about 28mg/kg or about 32mg/kg, for example, are infused intravenously on days 1, 7 days, 15 days, and 21 days (i.e.g., once per week), about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, A dose of about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, or about 32 mg/kg), or any increment thereof, and administering gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) to the subject on days 1, 8, and 15. In some examples, at 125mg/m 2 Intravenous administration to a subjectPaclitaxel is administered (e.g., by intravenous infusion). In some examples, at 1000mg/m 2 Gemcitabine is administered (e.g., intravenously infused) intravenously to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) may be reduced to 100mg/m 2 Or 75mg/m 2 . In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some embodiments, the methods described herein for treating a solid tumor (e.g., PDAC) comprise one or more treatment cycles of 28 days, wherein 0.2mg/kg to 0.5mg/kg, 0.5mg/kg to 1mg/kg, 1mg/kg to 2mg/kg, 3mg/kg to 4mg/kg, 4mg/kg to 8mg/kg, 4mg/kg to 6mg/kg, 4mg/kg to 6.3mg/kg, 6mg/kg to 8mg/kg, 6.3mg/kg to 8mg/kg, 8mg/kg to 12mg/kg, 8mg/kg to 10mg/kg, 10mg/kg to 12mg/kg, 12mg/kg to 16mg/kg, 16mg/kg to 20mg/kg, 20mg/kg to 24mg/kg, 24mg/kg to 28mg/kg, or 32 mg/for example, are infused intravenously on days 1, 7 days, 15 days, and 21 (i.e.g., once per week), about 0.2mg/kg, 0.5mg/kg, 0.6mg/kg, 0.63mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, about 6.3mg/kg, 7mg/kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 13mg/kg, A dose of 31mg/kg or 32 mg/kg) or any ascending dose within these ranges, and gemcitabine and paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) are administered to the subject on days 1, 8, and 15.
In some examples, at 125mg/m 2 Paclitaxel is administered intravenously (e.g., intravenous infusion) to the subject. In some examples, at 1000mg/m 2 Gemcitabine is administered (e.g., intravenously infused) intravenously to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) may be reduced to 100mg/m 2 Or 75mg/m 2 . In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some embodiments, the methods described herein for treating a solid tumor (e.g., PDAC) comprise one or more treatment cycles of 28 days, wherein about 0.2mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 1mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 6.3mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg or about 20mg/kg or any of these are administered via intravenous infusion via day 1, and wherein the anti-tumor particles are administered to the subject on day 1, e.g., paclitaxel, and the administration of the anti-tumor particles on day 1, e.g., 15, the administration of the particles is performed at an increment of paclitaxel to the subject. In some examples, at 125mg/m 2 Paclitaxel is administered intravenously (e.g., intravenous infusion) to the subject. In some examples, at 1000mg/m 2 Gemcitabine is administered (e.g., intravenously infused) intravenously to a subject. When required, the dose of gemcitabine may be reduced to 800mg/m 2 Or 600mg/m 2 . Alternatively or additionally, the dose of paclitaxel (e.g., nanoparticle albumin-bound paclitaxel) may be reduced to 100mg/m 2 Or 75mg/m 2 . In some embodiments, the anti-Gal 9 antibody can be administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week. For example, the anti-galectin-9 antibody is administered to the subject at a dose of 10mg/kg once a week or at a plateau dose of 650-700mg once a week. Alternatively, the anti-galectin-9 antibody is administered to the subject at a dose of 16mg/kg once a week or at a plateau dose of 1040-1120mg once a week.
In some cases, gal-9 antibody treatment can be initiated simultaneously with chemotherapy (e.g., gemcitabine and nab-paclitaxel). Alternatively, gal-9 antibody treatment may be initiated after a chemotherapy regimen (e.g., gemcitabine and nab-paclitaxel) has been initiated. In some cases, the Gal-9 antibody treatment is administered concurrently with chemotherapy (e.g., gemcitabine and nab-paclitaxel), followed by discontinuation of the chemotherapy. In some cases where chemotherapy is discontinued, the anti-Gal-9 antibody treatment regimen may be continued.
In any of the above embodiments, the interval or period may be weekly. In any of the above embodiments, the interval or period may be once every 2 weeks. In some embodiments, the regimen may be one cycle every 2 weeks, two cycles every 2 weeks, three cycles every 2 weeks, four cycles every 2 weeks, or more than four cycles every 2 weeks. In some embodiments, the treatment may be for 1 to 3 months once every 2 weeks, 3 to 6 months once every 2 weeks, 6 to 12 months once every 2 weeks, or 12 to 24 months or longer once every 2 weeks.
In any of the above embodiments, the interval or period may be 3 weeks. In some embodiments, the regimen may be one cycle every 3 weeks, two cycles every 3 weeks, three cycles every 3 weeks, four cycles every 3 weeks, or more than four cycles every 3 weeks. In some embodiments, the treatment may be for 1 to 3 months once every 3 weeks, 3 to 6 months once every 3 weeks, 6 to 12 months once every 3 weeks, or 12 to 24 months or more once every 3 weeks.
In any of the above embodiments, the interval or period may be 4 weeks or more. In some embodiments, the regimen is one cycle every 4 weeks or more, two cycles every 4 weeks or more, three cycles every 4 weeks or more, four cycles every 4 weeks or more, or more than four cycles every 4 or more. In some embodiments, the treatment may be for 1 to 3 months once every 4 weeks or more, 3 to 6 months once every 4 weeks or more, 6 to 12 months once every 4 weeks or more, or 12 to 24 months or more once every 4 weeks or more. In some embodiments, the treatment may be a combination of treatments at different times, e.g., a combination of 2 weeks, 3 weeks, 4 weeks, or more than 4 weeks. In some embodiments, the treatment interval may be adjusted according to the patient's response to the treatment. In some embodiments, the dose is adjusted according to the patient's response to treatment. In some embodiments, the dose varies between treatment intervals. In some embodiments, treatment may be temporarily stopped. In some embodiments, anti-galectin-9 therapy is temporarily discontinued. In some embodiments, chemotherapy is temporarily stopped. In some implementations, both are temporarily stopped.
In any of the combination treatment methods disclosed herein, one or more chemotherapeutic agents (e.g., gemcitabine and nab-paclitaxel) and an anti-galectin-9 antibody (e.g., G9.2-17IgG 4) may be administered on the same day. In this case, one or more chemotherapeutic agents may be administered to the subject prior to administration of the anti-galectin-9 antibody. In other examples, the subject may be administered one or more chemotherapeutic agents (e.g., gemcitabine and nab-paclitaxel) and an anti-galectin-9 antibody (e.g., G9.2-17IgG 4) for two consecutive days. The chemotherapeutic agent may be administered on the first day of treatment and the anti-galectin-9 antibody may be administered on the second day.
In other cases, checkpoint inhibitors, such as any of the chemotherapeutic agents disclosed herein (e.g., gemcitabine and nab-paclitaxel), may be administered about 1-7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) prior to administration of an anti-galectin 9 antibody disclosed herein, such as G9.2-17.
In some examples, an anti-galectin 9 antibody may be administered to a subject prior to administration of a chemotherapeutic agent. In other cases, administration of the anti-galectin 9 antibody and administration of the chemotherapeutic agent are performed for two consecutive days. The anti-galectin-9 antibody may be administered to the subject on the first day of administration, and the chemotherapeutic agent may be administered to the subject on a subsequent day. In other cases, an anti-galectin 9 antibody disclosed herein (such as G9.2-17) can be administered about 1-7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) prior to administration of a chemotherapeutic agent (such as gemcitabine and nab-paclitaxel).
In some embodiments, provided herein are methods in which an anti-gal-9 antibody is administered in combination with chemotherapy (e.g., gemcitabine and nab-paclitaxel) for improving overall response (e.g., at 3, 6, or 12 months), e.g., compared to baseline levels prior to initiation of treatment. In some embodiments, provided herein are methods for achieving a complete response, partial response, or stabilizing a disease (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time according to RECIST or irec criteria). Such a response may be temporary or permanent for a period of time. In some embodiments, the method may increase the likelihood of complete response, partial response, or stable disease (e.g., as measured at 3 months, 6 months, or 12 months), e.g., compared to baseline levels prior to initiation of treatment. Such a response may be temporary or permanent for a period of time.
In some embodiments, the treatment may result in a longer lifetime or greater likelihood of survival, e.g., at a certain time, e.g., at 6 or 12 months or at a later point in time.
In any of the methods described herein, partial response, stable disease, complete response, partial response, stable disease, progressive disease, disease progression (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time) can be assessed according to RECIST criteria or irec criteria. Responses to treatment (e.g., treatment of solid tumors as described herein) may be assessed according to RECIST or RECIST 1.1 criteria and/or irRC, irRECIST, iRECIST, imRECISTPDAC, as described in the following documents: eisenhower et al New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1); european Journal Of Cancer 45 (2009) 228-247; or Borcoman et al Annals of Oncology 30:385-396,2019; nishino et al, clin Cancer Res 2013;19 (14) 3936-3943, the contents of each of these documents are incorporated herein by reference in their entirety.
In some embodiments, described herein are methods of improving quality of life and symptom control (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time) compared to a baseline prior to initiation of treatment. In some embodiments, the improvement can be measured on an ECOG scale as described in example 2 herein.
In some embodiments, the present disclosure provides methods of reducing or maintaining tumor size (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time) in a subject (including a human subject) relative to a baseline tumor size in the subject prior to initiation of treatment, the methods comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments, the present disclosure provides methods for increasing the likelihood of reducing or maintaining tumor size in a subject (including a human subject), either permanently or within a minimum period of time (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time), e.g., as compared to a baseline level prior to initiation of treatment. In some embodiments, the present disclosure provides methods for reducing or maintaining tumor burden (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time) in a subject (including a human subject) as compared to a baseline level prior to initiation of treatment. Thus, in some embodiments, tumor size and/or burden is measured in regularly scheduled sub-fractionated scans (e.g., contrast-agent-bearing CT, contrast-agent-bearing MRI, PET-CT (diagnostic CT), and/or X-rays).
In some embodiments, provided herein are methods for increasing the time to disease progression or increasing the time to progression-free survival (e.g., as measured at 6 months), comprising administering an anti-gal-9 antibody in combination with chemotherapy (e.g., gemcitabine and nab-paclitaxel). In some embodiments, the method may result in a greater likelihood of progression free survival (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time after initiation of treatment). In some embodiments, provided herein are methods for improving the duration and depth of response according to RECIST 1.1 criteria (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time after initiation of treatment), comprising administering an anti-gal-9 antibody in combination with chemotherapy (e.g., gemcitabine and nab-paclitaxel).
In some embodiments, the methods provided herein, wherein an anti-gal-9 antibody is administered in combination with chemotherapy (e.g., gemcitabine and nab-paclitaxel), can improve quality of life and/or improve symptom control (e.g., as measured at 1 month, 3 months, 6 months, or 12 months, or at a later time using the ECOG scale) as compared to the baseline prior to the start.
Alternatively or additionally, anti-galectin-9 antibodies can be used in combination with a regimen comprising UGN-102, UGN-201, or UGN-302. In one embodiment, UGN-102, UGN-201, or UGN-302 is formulated in a hydrogel (e.g., a hydrogel based on inverse thermal gelation technology). In some examples, the anti-galectin-9 antibody can be administered prior to UGN-102, UGN-201, or UGN-302. In some examples, the anti-galectin-9 antibody can be administered concurrently with UGN-102, UGN-201, or UGN-302. In some examples, the anti-galectin-9 antibody can be administered subsequent to UGN-102, UGN-201, or UGN-302.
(iv)Monitoring treatment response
Patient response to any of the treatments disclosed herein can be monitored by conventional practice or as disclosed herein.
In some embodiments, the response to treatment can also be characterized by: immunophenotype in blood and tumor, cytokine profile (serum), soluble galectin 9 level in blood (serum or plasma), galectin 9 tumor tissue expression level and expression pattern obtained by immunohistochemistry (tumor, stroma, immune cells), tumor Mutational Burden (TMB), PDL-1 expression (e.g. by immunohistochemistry), mismatch repair status or tumor markers associated with disease (e.g. as measured at 3 months, 6 months or 12 months or later). Non-limiting examples of such tumor markers include Ca15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters can be compared to baseline levels prior to initiation of treatment.
In any of the methods disclosed herein, the subject may be examined for one or more of the following characteristics prior to, during, and/or after treatment: (a) One or more tumor markers in a blood sample from a subject, optionally wherein the one or more tumor markers comprise CA15-3, CA-125, CEA, CA19-9, and/or alpha-fetoprotein, and any other tumor type specific tumor markers; (b) cytokine profile; and (c) galectin 9 serum/plasma levels, d) peripheral blood mononuclear cell immunophenotyping, e) tumor tissue biopsy/resection specimen multiplex immunophenotyping, f) tumor tissue biopsy/resection specimen galectin 9 expression levels and patterns, g) any other immunophenotyping test such as: PDL-1 immunohistochemistry, tumor Mutational Burden (TMB), tumor microsatellite instability status, and groups such as: in NanoStringGene expression system, 18-gene marker and PanCancer IO 360 TM Assay (NanoString Technologies) et al>HalioDx, immunoSeq-Adaptive Biotechnologies, TIS. Other suitable biomarkers specific for a target tumor, such as PDAC, may also be used.
Thus, in some embodiments, methods of modulating a therapeutic condition are contemplated herein based on one or more features disclosed herein.
In some embodiments, an increase in the overall immune response, e.g., an increase in the overall inflammatory immune response, is determined by a decrease in tumor weight, tumor size, or tumor burden, or any RECIST criteria described herein. In some embodiments, the increase in the overall immune response is determined by an increase in the level of one or more pro-inflammatory cytokines (e.g., including two or more, three or more) or a majority of pro-inflammatory cytokines (one or more, two or more or a majority of anti-inflammatory and/or immunosuppressive cytokines and/or one or more of the most potent anti-inflammatory or immunosuppressive cytokines is reduced or held constant). In some embodiments, the increase in the overall immune response is determined by an increased level of one or more of the most potent pro-inflammatory cytokines (one or more anti-inflammatory and/or immunosuppressive cytokines including one or more of the most potent cytokines decrease or remain unchanged). In some embodiments, the increase in the overall immune response is determined by a reduced level of one or more including a majority of immunosuppression and/or anti-inflammatory cytokines (the level of one or more or most of pro-inflammatory cytokines including, for example, the most potent pro-inflammatory cytokines increases or remains unchanged). In some embodiments, the increase in the overall immune response is determined by the increased level of one or more of the most potent anti-inflammatory and/or immunosuppressive cytokines (one or more or most pro-inflammatory cytokines including, for example, the most potent pro-inflammatory cytokines are increased or remain unchanged). In some embodiments, the increase in the overall immune response is determined by a combination of any of the above. Furthermore, an increase (or up-regulation) of one type of immune response parameter may result in a corresponding decrease (or down-regulation) of another type of immune response parameter. For example, an increase in the production of certain pro-inflammatory cytokines may result in the down-regulation of certain anti-inflammatory and/or immunosuppressive cytokines, and vice versa.
(D) Modulating immune responses
In some embodiments, the methods described herein (wherein the Gal-9 antibody is administered with chemotherapy such as gemcitabine and nab-paclitaxel) can modulate the levels of immune cells and immune cell markers in blood or tumors. Such changes may be measured in patient blood and tissue samples using methods known in the art, such as multiple flow cytometry and multiple immunohistochemistry. For example, a set of phenotypic and functional PBMC immunomarkers may be evaluated at baseline before the start of treatment and at different time points during treatment. Table 2 lists non-limiting examples of markers that can be used in these assessment methods. Flow Cytometry (FC) is a rapid and informative first-choice technique for analyzing cellular phenotypes and functions, and has been prominent in immunophenotyping. It allows the characterization of many cell subsets, including rare subsets, in complex mixtures (such as blood) and represents a rapid method of acquiring large amounts of data. The FC has the advantages of high speed, high sensitivity and strong specificity. Standardized antibody panels and procedures can be used to analyze and classify immune cell subtypes. Multiplex IHC is a powerful investigation tool that provides objective quantitative data describing the number and location of immune subpopulations in the tumor immune environment and allows multiple markers to be assessed on a single tissue section. Computer algorithms can be used to quantify IHC-based biomarker levels from whole slide (slide) images of patient biopsies, combining chromogenic IHC methods and staining with digital pathology methods.
TABLE 2 PBMC typing markers
In some embodiments, the present disclosure provides methods for modulating an immune response (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time) in a subject (including a human subject), the methods comprising administering to the subject a therapeutically effective amount of an anti-galectin-9 antibody as disclosed herein. In some embodiments, the present disclosure provides methods for modulating the levels of immune cells and immune cell markers (including but not limited to those described herein in table 2) in the blood or in tumors of a subject (including a human subject), e.g., compared to baseline levels prior to initiation of treatment, comprising administering to the subject a therapeutically effective amount of an anti-galectin 9 antibody as disclosed herein. In some embodiments, the overall result of modulation is up-regulation of pro-inflammatory immune cells and/or down-regulation of immunosuppressive immune cells.
Thus, in some embodiments, the methods described herein (wherein an anti-gal 9 antibody is administered in combination with chemotherapy) can modulate immune activation markers, such as those in table 2. In some embodiments, the methods described herein (wherein the anti-gal 9 antibody is administered alone or in combination with a checkpoint inhibitor therapy) result in one or more of the following: (1) an increase in more CD8 cells in the plasma or tumor tissue, (2) a decrease in T regulatory cells (tregs) in the plasma or tumor tissue, (3) an increase in M1 macrophages in the plasma or tumor tissue, and (4) a decrease in MDSCs in the plasma or tumor tissue, and (5) a decrease in M2 macrophages in the plasma or tumor tissue (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time). In some embodiments, the marker assessed using the techniques described above or known in the art is selected from the group consisting of CD4, CD8, CD14, CD11b/c and CD25. These parameters can be compared to baseline levels prior to initiation of treatment.
In some embodiments, the treatment as described herein results in a change in pro-inflammatory and anti-inflammatory cytokines. In some embodiments, provided herein are methods for one or more of the following: (1) increasing the level of ifnγ in plasma or tumor tissue; (2) increasing the level of tnfα in plasma or tumor tissue; (3) Lowering the level of IL-10 in plasma or tumor tissue (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time). These parameters can be compared to baseline levels prior to initiation of treatment.
In some embodiments, changes in cytokines or immune cells can be assessed between 1 tumor biopsy prior to dose and repeated biopsies taken at a viable time. In some embodiments, the change in cytokines or immune cells can be assessed between duplicate biopsies. In some embodiments, described herein are methods of modulating the level of soluble galectin 9 in blood (serum or plasma) or the level of galectin 9 tumor tissue expression and the level of one or more of the expression patterns obtained by immunohistochemistry (tumor, stroma, immune cells) (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time). In some embodiments, described herein are methods for reducing the level of soluble galectin 9 in blood (serum or plasma) or the level of galectin 9 tumor tissue expression and the level of one or more of the expression patterns (tumor, matrix, immune cells) obtained by immunohistochemical reduction (e.g., as measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or at a later time). These galectin 9 levels can be compared to baseline levels prior to initiation of treatment. In some embodiments, the measurement is performed at 2 months.
In some embodiments, described herein are methods for modulating PD-L1 expression levels, e.g., as assessed by immunohistochemistry. In some embodiments, the disclosure provides methods for modulating PDL-1 expression (e.g., as assessed by immunohistochemistry) (e.g., as measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or at a later time), comprising administering to a subject a therapeutically effective amount of an anti-galectin 9 antibody as disclosed herein. In some embodiments of the methods, PDL-1 expression (e.g., as assessed by immunohistochemistry) remains unchanged. PD-L1 levels can be compared to baseline levels prior to initiation of treatment. In some embodiments, the methods provided herein reduce PDL-1 expression, e.g., as assessed by immunohistochemistry. The PD-L1 level may be measured using conventional methods known in the art. In one non-limiting example, PD-L1 (SP 263) can be used to detect PD-L1 in cancer tissue using immunohistochemistry. In some embodiments, provided herein are methods for modulating (increasing or decreasing) one or more tumor markers associated with a disease (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time). Non-limiting examples of such tumor markers include Ca15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters can be compared to baseline levels prior to initiation of treatment. In some embodiments, the methods provided herein reduce the occurrence of one or more tumor markers associated with a disease.
In some embodiments, the disclosure provides methods for modulating PDL-1 expression (e.g., as assessed by immunohistochemistry) (e.g., as measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or at a later time), comprising administering to a subject a therapeutically effective amount of an anti-galectin 9 antibody as disclosed herein. In some embodiments of the methods, PDL-1 expression (e.g., as assessed by immunohistochemistry) remains unchanged. PD-L1 levels can be compared to baseline levels prior to initiation of treatment. In some embodiments, the methods provided herein reduce PDL-1 expression, e.g., as assessed by immunohistochemistry.
In some embodiments, the disclosure provides methods for modulating (increasing or decreasing) one or more biomarkers associated with a disease (e.g., as measured at 2 weeks, 4 weeks, 1 month, 3 months, 6 months, or 12 months, or at a later time), comprising administering to a subject a therapeutically effective amount of an anti-galectin 9 antibody as disclosed herein. Conventional methods may be used to measure the level of a biomarker in clinical tissue from a patient, such as multiple immunofluorescence (mIF) techniques, as described in the examples herein. One exemplary set of biomarkers may include CD3, CD4, CD8, CD45RO, foxP3, CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, arginase 1, granzyme B, ki67, PD1, PD-L1, F4/80, ly6G/C and PanCK.
These markers can be compared to baseline levels prior to initiation of treatment (e.g., at intervals, for example, at 3 months, 6 months, or 12 months). In some embodiments, the cytokine profile is modulated.
In some embodiments, the present disclosure provides methods of modulating an immune response in a subject. The immune response may be a T cell-mediated and/or B cell-mediated immune response that is affected by modulation of immune cell activity, e.g., T cell activation. In one embodiment of the present disclosure, the immune response is T cell mediated. As used herein, the term "modulate" means a change or modification, and includes up-and down-regulation. For example, "modulating an immune response" means changing or altering the state of one or more immune response parameters. Exemplary parameters of a T cell mediated immune response include the level of T cells (e.g., an increase or decrease in effector T cells) and the level of T cell activation (e.g., an increase or decrease in the production of certain cytokines). Exemplary parameters of a B cell mediated immune response include an increase in B cell levels, B cell activation, and B cell mediated antibody production.
When the immune response is modulated, some parameters of the immune response may decrease, while others may increase. For example, in some cases, modulating the immune response causes an increase (or up-regulation) in one or more immune response parameters and a decrease (or down-regulation) in one or more other immune response parameters, and the result is an overall increase in the immune response, e.g., an overall increase in the inflammatory immune response. In another example, modulating the immune response causes an increase (or up-regulation) in one or more immune response parameters and a decrease (or down-regulation) in one or more other immune response parameters, and the result is an overall decrease in the immune response, e.g., an overall decrease in inflammatory response.
In some embodiments, provided herein are methods for modulating soluble galectin-9 levels or galectin-9 tumor tissue expression levels and immunohistochemically derived expression patterns (tumor, matrix, immune cells) in blood (serum or plasma) in a subject (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time) comprising administering an anti-gal 9 antibody in combination with chemotherapy. Galectin 9 levels in the subject can be compared to baseline levels prior to initiation of treatment.
In some embodiments, provided herein are methods for reducing the level of soluble galectin 9 in blood (serum or plasma) or the level of galectin 9 tumor tissue expression and the level of one or more of the expression patterns (tumor, matrix, immune cells) obtained by immunohistochemical reduction (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time).
In some embodiments, provided herein are methods (including administration of an anti-gal 9 antibody in combination with chemotherapy) for modulating one or more tumor markers (increase or decrease) associated with a disease (e.g., as measured at 3 months, 6 months, or 12 months, or at a later time). Non-limiting examples of such tumor markers include Ca15-3, CA-125, CEA, CA19-9, alpha-fetoprotein. These parameters can be compared to baseline levels prior to initiation of treatment.
Kit for use in combination therapy of solid tumors
The present disclosure also provides kits for use in treating or alleviating solid tumors, such as PDA, CRC, HCC or cholangiocarcinoma, and other solid tumors described herein. Such kits may include one or more containers comprising an anti-galectin-9 antibody, such as any of those described herein (e.g., G9.2-17 (IgG 4)), and optionally one or more chemotherapeutic agents (e.g., gemcitabine and/or paclitaxel) to be used with the anti-galectin-9 antibodies described herein, also described herein.
In some embodiments, the kit may include instructions for use according to any of the methods described herein. Included instructions may include descriptions of administration of an anti-galectin 9 antibody and one or more chemotherapeutic agents to treat, delay onset, or reduce target diseases such as those described herein. In some embodiments, the kit further comprises a description of selecting an individual suitable for treatment based on identifying whether the individual has the target disease (e.g., applying a diagnostic method as described herein). In other embodiments, the instructions comprise a description of administering the antibody to an individual at risk of developing the target disease.
Instructions relating to the use of anti-galectin 9 antibodies and one or more chemotherapeutic agents typically include information about the dosage, dosing regimen, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disc) are also acceptable.
The label or package insert indicates that the composition is for use in treating, delaying onset, and/or alleviating a solid tumor. In some embodiments, instructions for practicing any of the methods described herein are provided.
The kits of the invention are in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed mylar or plastic bags), and the like. Packages for use with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or infusion devices (e.g., micropumps), are also contemplated. In some embodiments, the kit has a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (e.g., the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-galectin-9 antibody as described herein.
The kit may optionally provide additional components such as buffers and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the above-described kit.
General technique
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in documents such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. Gait, 1984); methods in Molecular Biology, humana Press; cell Biology A Laboratory Notebook (J.E.Cellis, 1998) Academic Press; animal Cell Culture (r.i. freshney, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; cell and Tissue Culture: laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell, 1993-8) J.Wiley and Sons; methods in Enzymology (Academic Press, inc.); handbook of Experimental Immunology (d.m. weir and c.c. blackwell); gene Transfer Vectors for Mammalian Cells (J.M.Miller and M.P.Calos. Ed., 1987); current Protocols in Molecular Biology (F.M. Ausubel et al, 1987); PCR: the Polymerase Chain Reaction, (Mullis et al, 1994); current Protocols in Immunology (J.E. Coligan et al, 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.convers, 1997); antibodies (P.Finch, 1997); antibodies a practical approach (D.Catty. Eds., IRL Press, 1988-1989); monoclonal antibodies: a practical approach (P.shepherd and C.dean, eds., (Oxford University Press, 2000)); using anti-ibodies a laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); the Antibodies (M.Zanetti and J.D.Capra, eds., harwood Academic Publishers, 1995).
Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. For the purposes or subject matter recited herein, all publications recited herein are incorporated by reference.
Examples
While the present disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of this disclosure.
Example 1In vivo studies of anti-galectin-9 antibodies in combination with chemotherapeutic agents for cancer treatment in pancreatic cancer mouse models
Anti-galectin-9 IgG4 fully human antibodies (G9.2-17 (IgG 4)) were performed in a mouse model of pancreatic cancer-as a single agent or in combination with other systemic chemotherapeutic anti-cancer forms-for preclinical evaluation of treatment of refractory solid tumors.
The specific animal used was an orthotopic mPA6115 pancreatic cancer xenograft model in female C57BL/6 mice. To generate this model, first, tumors were derived from the mPA6115 mouse, which is a mouse allograft model of Pancreatic Ductal Adenocarcinoma (PDAC) that retains morphological similarity to human PDAC. mPA6115 mouse strain carrying conditional mutant (Kras LSL-G12D/WT ) Constitutive deletions of Trp53 (P53 KO/KO) and Cre driven by the Pdx1 gene promoter, and develop into severe PDAC tumors at 8 weeks of age.
At this point, mPA6115 mice with accessible tumors were sacrificed and their pancreatic tumors were collected. The collected tumor tissue was cut into small pieces (-2 mm) 3 ) And Subcutaneously (SC) transplanted into isogenic receptor C57BL/6 mice. Maintaining the seed tumor under the skin of the C57BL/6 mouse until the seed tumor volume reaches 700-1000 mm 3 . Once the seed tumor reaches the desired volume, the tumor is harvested and cut to a diameter of about 2mm 3 Is a block of (c). The tumors were then washed with ice-cold Roswell Park Memorial Institute (RPMI) 1640 medium (serum-free) to remove adjacent non-tumor tissue. The tumor mass was then placed in ice-cold RPMI 1640 medium until implantation in situ. On the same day as seed tumors were collected, 6-7 week old female C57BL/6 mice were pancreas transplanted in situ. Specifically, after complete anesthesia of the animals, a small longitudinal incision was made under the left lower rib to expose the spleen and pancreas under the spleen. One seed tumor mass per mouse was sutured with 6-0 silk threads Into the pancreas. The tissue surrounding the tumor mass was then sutured with 6-0 silk threads and the tumor mass was wrapped with pancreatic tissue. The abdomen was then sutured with 4-0 silk threads. Following tumor implantation, the animals were housed in warm cages and subsequently returned to the animal room after complete anesthesia was awakened.
On the day of implantation, the implanted mice were randomized into 6 groups based on their body weight, with randomization based on the "matching distribution" method (study directortm software, version 3.1.399.19). The date of the random grouping is indicated as day 0. Three days after implantation, animals began the dosing regimen according to group number. The dosing regimen for each group is provided in table 3 below.
TABLE 3 study dosing regimen
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P. = intraperitoneal; v. = intravenous; QW = once weekly; q4d=once every four days
For these studies, anti-galectin-9 mouse IgG1 was used. This antibody, known as the anti-Gal 9 mAb, is a mouse IgG1 version of the human G9.2-17 antibody, which binds to the carbohydrate binding domain 2 (CRD 2) of galectin-9, which is identical to G9.2-17, and has the same VH and VL regions as G9.2-17. Thus, the data obtained using the anti-Gal 9 mAb correlated with human efficacy of G9.2-17. In addition to mice treated with only anti-Gal 9 mAb (group 4), implanted mice of groups 5 and 6 were treated with standard of care chemotherapy (gemcitabine/abraxane regimen) or a combination of anti-Gal 9 mAb and chemotherapy.
Group 1-7 mice were examined daily for morbidity and mortality following pancreas in situ transplantation. During routine monitoring, animals were examined for any effect of tumor growth and treatment on behavior, such as motility, food and water consumption, weight gain/loss, eye dullness/hair knot and any other abnormalities. Using a StudiDirector TM The software (version 3.1.399.19) measures body weight and tumor volume twice weekly after randomization. Measurements and monitoring were collected as described from day 0 until day 66 when the last mouse death was found. Blood, plasma, spleen and tumor were collected at the end of each mouse life. Table 4 below shows the average life of the mice by experimental group. The longest survival time for all control groups (groups 1, 2 and 3) was 33 days, while the last mice of group 4 (anti-galectin-9 IgG 1), group 5 (gemcitabine/abraxane), group 6 (combination therapy) died on days 55, 41 and 66, respectively.
TABLE 4 average longevity of mice in each group
The primary survival endpoint of animals transplanted with in situ KPC tumors was assessed by estimating the survival curves of each group considered separately using the Kaplan-Meier method and making statistical comparisons using a log rank test. Specifically, the Kaplan-Meier survival curve/log rank test (SPSS 18) was used. Kaplan-Meier survival curves and log rank test are shown in fig. 1A-1D. The results of the log rank test are provided in table 5.
TABLE 5 logarithmic rank test
*，p<0.05；**，p<0.01；***，p<0.001；ns，p≥0.05
The risk ratio (HR) and its 95% confidence interval (% 95 CI) for groups 4-6 relative to groups 1, 2 and 3, respectively, were calculated using Cox regression analysis (coxph function of surviving R packets). We also used cox regression analysis to calculate the risk ratio (HR) of groups 5 and 6 to 4 and their 95% confidence interval (% 95 CI). Finally, we used cox regression analysis to calculate the risk ratio (HR) of groups 6 and 5 and their 95% confidence interval (% 95 CI). The results of the cox regression analysis are shown in fig. 2 and table 6.
TABLE 6 Cox regression analysis
Group of Comparison of p value HR HR(95％CI)
Group 4 Group 1 0.017 0.348 (0.146，0.83)
Group 5 Group 1 0.262 0.624 (0.274，1.422)
Group 6 Group 1 0.015 0.336 (0.14，0.806)
Group 4 Group 2 0.052 0.427 (0.181，1.009)
Group 5 Group 2 0.525 0.767 (0.339，1.738)
Group 6 Group 2 0.045 0.413 (0.173，0.982)
Group 4 Group 3 0.091 0.477 (0.202，1.125)
Group 5 Group 3 0.707 0.856 (0.379，1.932)
Group 6 Group 3 0.079 0.460 (0.193，1.094)
Group 5 Group 4 0.166 1.795 (0.784，4.109)
Group 6 Group 4 0.933 0.966 (0.424，2.198)
Group 6 Group 5 0.149 0.538 (0.232，1.248)
Group 2 Group 1 0.615 0.813 (0.364，1.818)
Group 3 Group 1 0.445 0.730 (0.325，1.639)
Group 3 Group 2 0.790 0.897 (0.402，2.002)
*，p<0.05；**，p<0.01；***，p<0.001；ns，p≥0.05
For the cox regression analysis using group 1 as a reference, the risk ratio of groups 4 and 6 was significantly lower than that of group 1, while the risk ratio of groups 2 and 3 was not significantly different from that of group 1. In the cox regression analysis using group 2 as a reference, the risk ratio of group 6 was significantly lower than that of group 2; however, the risk ratio of group 3 to group 2 is not significantly different. For the cox regression analysis using group 3 as a reference, the risk ratios of groups 4, 5 and 6 were not significantly different from group 3. In the cox regression analysis using group 4 as a reference, the risk ratio of groups 5 and 6 was not significantly different from that of group 4. Finally, cox regression analysis using group 5 as a reference showed that the risk ratio of group 6 was not significantly different from group 5.
These data demonstrate that the combination of anti-galectin-9 antibody and gemcitabine/abraxane is well tolerated, can be administered for extended periods of time (up to 16 doses for anti-galectin-9 IgG1 antibody (mouse IgG1 version) and 10 doses for gemcitabine/abraxane) and provides survival benefits compared to untreated animals (group 6 versus group 1: cox assay, hr=0.336, HR (95% ci) = (0.14,0.806), p=0.015, and p=0.051 LogRank test for average survival). The anti-galectin-9 IgG1 alone provided survival benefits compared to untreated animals (group 4 versus group 1: cox analysis, hr=0.348, HR (95% ci) = (0.146,0.83), p=0.017).
The last mouse in group 6 was found to die on day 66, and no tumor was found in the pancreas of the mice, thus terminating the study. According to historical data, the in situ mPA6115 model was employed at 100% in the vehicle group. Thus, the last mouse in group 6 was a complete responder to the anti-galectin-9/gemcitabine/abraxane combination regimen.
Body weight of mice transplanted with in situ KPC tumors was measured twice weekly after implantation/randomization (day 0) until all mice were euthanized Or die. FIG. 3 shows the use of a StudiDirector TM Weight measurements collected over the duration of the study period were measured by software (version 3.1.399.19). The last mouse in group 4 moribund and euthanized on day 55, with a tumor weight of 2544.6mg (tv= 1877.07mm 3 ). From day 51 to day 55, the body weight of only the last mouse in group 4 varied from 12.36% to-2.25% compared to the body weight of the first day of treatment. Weight loss is most likely associated with disorders caused by tumor growth.
Taken together, the data in this example demonstrate the safety and efficacy of the anti-galectin-9 regimen and the anti-galectin-9/gemcitabine/abraxane combination regimen in the orthotopic pancreatic cancer xenograft model mPA 6115.
Example 2: 1/2-phase open-label multicenter study of safety, pharmacokinetics and anti-tumor Activity of anti-galectin 9 monoclonal antibodies alone and in combination with chemotherapy in patients with metastatic solid tumors
Galectin-9 is a molecule that is overexpressed by many solid tumors, including pancreatic, colorectal, and hepatocellular carcinoma. Furthermore, galectin-9 is expressed on tumor-associated macrophages as well as on intratumoral immunosuppressive γδ T cells, thereby acting as an effective mediator of cancer-associated immunosuppression.
G9.2-17 (IgG 4) is a fully human IgG4 monoclonal antibody (mAb) targeting the galectin-9 (gal-9) protein. Gal-9 acts as an immunosuppressant, conferring tumor cell immune privileges and disabling immune-mediated cancer attack by modulating the sensitivity of macrophages, T cells, myeloid-derived suppressor cells, and cancer cells to cytotoxic T cell-induced death. Based on available data, blocking gal-9 by G9.2-17 (IgG 4) would interfere with the immunosuppressive function of gal-9, resulting in effective immune activation and tumor growth inhibition in multiple preclinical models.
Gal-9 can be overexpressed and/or secreted in many solid tumor types, including pancreatic adenocarcinoma, cholangiocarcinoma (CCA), colorectal cancer (CRC), breast cancer, bladder cancer, ovarian cancer, non-small and small cell lung cancer, nasopharyngeal cancer, malignant melanoma, ovarian cancer, etc., and high levels of tissue and/or circulating Gal-9 are associated with invasive tumor characteristics and poor survival outcomes.
Thus, the target indication for G9.2-17 (IgG 4) is recurrent or refractory metastatic solid tumors, where G9.2-17 (IgG 4) can be studied both as a single agent and in combination with a checkpoint inhibitor (programmed cell death 1[ pd 1] antibody).
Dose escalation (part 1) was performed in all com solid tumor types in order to establish the safety and tolerability profile of G9.2-17 (IgG 4), evaluate their immunogenic potential, establish Pharmacokinetic (PK) and Pharmacodynamic (PD) profiles, and reach recommended phase 2 doses (RP 2D). This may be the Maximum Tolerated Dose (MTD). The extended group (section 2) is intended to: first line metastatic Pancreatic Ductal Adenocarcinoma (PDAC), combined with gemcitabine/nab-paclitaxel; and CRC and CCA, e.g., as a single agent.
No other therapies targeting gal-9 are currently approved or in clinical trials for any indication.
In the non-clinical studies conducted so far, no significant toxicity was observed at doses about 500 times higher than those expected for human administration. Furthermore, G9.2-17 (IgG 4) has been shown to be highly specific for gal-9 and has been shown to be effective in a variety of animal models of cancer. The target patient population into the group was at advanced disease and failed in standard-of-care treatment prior to the study into the group. It is expected that G9.2-17 (IgG 4), alone or in combination with one or more of the chemotherapeutic agents disclosed herein, such as gemcitabine and paclitaxel, would be beneficial in the treatment of malignant tumors, such as malignant solid tumors.
Target and endpoint
Part 1: dose escalation
Part 2: group extension
Study design
This is an open-label, non-control, multicenter phase 1/2 study with up-dosing phase (part 1) and group expansion phase (part 2) in patients with recurrent/refractory metastatic solid tumors. This study was conducted in up to 20 study centers in the united states. The duration of the study was estimated to be 12-24 months. Survival follow-up lasted for up to 2 years. The study protocol is presented in fig. 4.
Duration of treatment and treatment period
Duration of treatment
Study drug administration was continued until disease progression, unacceptable toxicity occurred, or study was withdrawn. Patients who discontinued study medication prior to disease progression and were not treated with other systemic anti-cancer therapies will receive study follow-up until disease progression.
Treatment period
The study consisted of the following phases in part 1 and part 2:
screening period: up to 4 weeks (day-28 to day-1) before the first dose
Treatment period: 28-day treatment cycle as presented in the evaluation schedule (SoA; tables 13 and 14)
Late treatment stage: 30 days after the last treatment (treatment end visit/early termination visit)
Follow-up period: patients who have discontinued treatment for reasons other than disease progression and have not received additional systemic anti-cancer treatment are subjected to long-term follow-up for up to 2 years (visit once every 3 months)
Part 1: dose escalation stage
Dose discovery studies were performed using the Continuous Reassessment Method (CRM) (O' Quigley et al, 1990) to establish DLT and RP2D. Two to six patients per treatment group 1-6 were assigned G9.2-17 (IgG 4) to receive higher IV injections every two weeks (Q2W) on days 1 and 15 of each 28 day cycle, starting with a dose of 0.2 mg/kg. Patients assigned to a particular dose escalation group receive the corresponding study dose for that group. They received study medication at one of 8 dose levels until disease progressed, unacceptable toxicity occurred, or the study was exited for other reasons. Patients who have been withdrawn for reasons other than toxicity or tolerability problems during the first treatment cycle are replaced only.
For groups 1-6, two patients were dosed at a time according to the CRM design. Dose escalation is based on analysis of patient safety data, focusing on the occurrence of DLT at previous dose levels and other relevant safety and dosing data in previous cohorts. The dose escalation may occur after at least 28 days (1 cycle). Skipping of dose levels is not allowed.
After group 6 was completed according to the CRM design, weekly (QW) G9.2-17 (IgG 4) dosing regimens were evaluated, provided RP2D had not been reached in the CRM design. Groups 7 and 8 were not evaluated with the CRM design. Once DLT was not identified, only patients were allowed to enter group 7.
For groups 7 and 8, four patients were dosed once per group. Four patients at each dose level in groups 7 and 8 were assigned G9.2-17 (IGG 4) that received higher IV injections weekly (QW) on days 1, 8, 15, and 22 of each 28 day cycle. Starting from the first four patients in group 7, dose escalation to the next group only occurs without identification of DLT. If a single DLT is recorded in group 7, no other patients are dosed within the group and group 8 is not activated.
Part 1, groups 1-8, were included in approximately 36 patients. A total of 6 dose levels were evaluated in the CRM design:
dose escalation cohort 1=0.2 mg/kg Q2W
Dose escalation cohort 2=0.63 mg/kg Q2W
Dose escalation cohort 3=2 mg/kg Q2W
Dose escalation cohort 4=6.3 mg/kg Q2W
Dose escalation cohort 5=10 mg/kg Q2W
Dose escalation cohort 6=16 mg/kg Q2W
Consider RP2D, which includes another 2 dose levels:
dose escalation cohort 7=10 mg/kg QW
Dose escalation cohort 8=16 mg/kg QW
Patients treated in early groups prior to identification of RP2D were allowed to dose escalation up to the highest dose level cleared. After a complete cycle, dose escalation may occur after at least 28 days (1 cycle). The dose escalation may not occur in the middle of the cycle. Patients may continue to dose escalation to the highest approved dose level until they discontinue due to toxicity or disease progression or for other reasons (e.g., patient selection discontinues the study).
The dose escalation is based on DLT production in patients treated at previous dose levels. For each dose group, a priori DLT probabilities were specified based on GLP-compliant toxicity studies and preclinical models. For a prescribed target DLT rate and total number of dose levels, a skeleton of the power model d ∈p (a) is generated according to the methods of Lee and Cheung using a priori MTD at the median dose level adjusted by PK/PD data and a spacing metric of δ=0.05 (Lee and Cheung, 2011). The prior distribution of parameter "a" has a normal distribution with zero mean, with minimal prior variance information. If the lower limit of the Agrest and Coull two Confidence Intervals (CI) for the lowest study dose level exceeds the target DLT rate, the trial is stopped for safety considerations (Agrest and Coull, 1998). RP2D is the MTD dose derived from part 1.
If DLT occurs in any patient during the first 28 days of treatment, administration of the study drug to that patient is permanently discontinued.
For patients experiencing toxicity outside the DLT window, including IMAR, dose reduction is only allowed if clinical benefit is expected and clinical benefit can continue to be obtained with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50%, and possibly further reduced by 50%, as defined by the dose modification guidelines provided in table 7. No further dose reduction is allowed.
Table 7. Recommended dose modification of G9.2-17 (IgG 4) (AE outside DLT Window and outside IMAR)
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Part 1 completion
Part 1 is complete when up to six patients have received doses identified as RP 2D. RP2D is based in part on continuous re-assessment method (CRM) study design, PK and PD data parameters, additional safety and efficacy data, and any other factors to be considered.
Backfill group
The purpose of the backfill group was to assess the safety, tolerability and biological effects of G9.2-17 (IGG 4) in patients with tumors positive for gal-9. The gal-9 state of the RP2D group is determined retrospectively. If fewer than 6 gal-9 positive tumor patients were treated with RP2D, patients designated for backfill groups required prospective assessment of gal-9 tumor status by IHC. Additional patients with up to 6 additional tumors positive for gal-9 may be enrolled in the backfill group at RP2D dose levels.
Part 2: group extension phase
The second part of the protocol was designed with a Simon two-stage optimization and included approximately 223 patients. Groups of extended PDACs, CRCs, and CCA and/or potentially other solid tumor types are planned, based on tumor-specific considerations implemented for the extended group and clinical trial endpoints. The rationale behind this approach is to ensure the feasibility of recruitment, as well as capture the clinical needs of a particular indication.
CRC and CCA patients received one of two treatments (4 treatment groups total):
g9.2-17 (IgG 4) as a single agent
PDAC patients received G9.2-17 (IGG 4) in combination with gemcitabine/nab-paclitaxel.
For all patients receiving combination therapy (gemcitabine/nab-paclitaxel + G9.2-17 (IgG 4)), the therapy may be administered on the same day. Gemcitabine/nab-paclitaxel should be administered before G9.2-17 (IgG 4). If the same day of administration cannot be completed for any reason, gemcitabine/nab-paclitaxel should be administered on the first day and G9.2-17 (IgG 4) on the subsequent day.
CRC and CCA patients
Treatment of a single agent group or a combined agent group of CRC and CCA patients may be performed in parallel.
G9.2-17 (IgG 4) monotherapy
The initial dose of G9.2-17 (IgG 4) in monotherapy was RP2D identified in section 1. For the CRC and CCA monotherapy groups, an optimized two-stage design (stages I and II) was used to examine the null hypothesis for ORR 3.ltoreq.5% with the alternative hypothesis for ORR 3.gtoreq.15% within the single agent group.
After testing 23 patients for study drug in stage I, the trial group was terminated if 1 patient responded. If the trial proceeds to phase II of Simon optimization design, approximately 33 patients are additionally treated in each of the single agent groups. If the total number of response patients is less than or equal to 5, the study medication in the group is rejected. If ≡6 patients have confirmed ORR3, part 3 extension cohort of the group is activated and described in protocol revision.
Dose reduction is only allowed if clinical benefit is expected and clinical benefit can continue to be expected with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50%, and possibly further reduced by 50%, as defined by the dose modification guidelines provided in this protocol. No further dose reduction is allowed.
PDAC patient
Part 2 group of metastatic PDAC patients require combination therapy of G9.2-17 (IgG 4) and gemcitabine/nab-paclitaxel in a first line metastatic setting.
The dose of G9.2-17 (IgG 4) was the RP2D-1 dose, which was the dose immediately preceding the RP2D dose identified in section 1. To ensure patient safety, a safe break-in was performed, wherein the first 8 patients were dosed and the group was continued only if less than or equal to 2 patients developed DLT (below 25% Target Toxicity Level (TTL)). If 3 or more patients develop DLT, the combination treatment group is terminated. In this combination treatment abrasion group, only patients who were withdrawn for reasons other than toxicity or tolerability problems during the first treatment cycle were replaced. If DLT occurs in any of the 8 safely-running-in patients during the first 28 days of treatment, administration of the study drug to that patient is permanently discontinued.
For patients experiencing toxicity outside the DLT window, dose reduction is allowed when clinical benefit is expected and clinical benefit can continue to be obtained with lower doses of G9.2-17 (IgG 4). The dose of G9.2-17 (IgG 4) was initially reduced by 50% and possibly by a further 50%. No further dose reduction is allowed. Allows for dose adjustment of gemcitabine and/or nab-paclitaxel.
If IMAR onset/recurrence cannot be managed by reducing the dose of either agent, both study medications must be discontinued.
The primary efficacy endpoint was patient PFS6. In a first line metastatic environment using gemcitabine/nab-paclitaxel, PFS6 was reported to be 50% (von Hoff et al 2013). After testing the 11 patients in the G9.2-17 (IGG 4)/chemotherapy combination in the first stage of the Simon two-stage design, the trial was terminated if 6 or less patients exhibited PFS for > 6 months. If the trial proceeds to the second phase of the Simon two-phase design, about 14 patients receive additional treatment. If the total number of responsive patients for PFS-6 is less than or equal to 16, the study group is rejected.
Part 2 completion
The completion of part 2 depends on the patient ORR 3 of the CRC and CCA patients and the PFS6 of the PDAC.
Part 3: expansion of
If a promising efficacy signal attributable to a tumor type is identified in one or more of the test groups, an expanded group is initiated to confirm the findings as described above. The sample size for each extension group is determined based on the point estimate determined in section 2 and a predetermined level of accuracy of 95% CI around ORR/OS and PFS. Prior to initiating section 3, a protocol revision is submitted including detailed information about the expanded population, treatment protocol, and statistical analysis plan.
Dose limiting toxicity criteria
Dose limiting toxicity assessed in this trial was defined as clinically significant hematological and/or non-hematological AE or abnormal laboratory values that were assessed as not associated with metastatic tumor disease progression, concomitant disease or concomitant medication, and possibly associated with or correlated with study drug, and occurred during the first cycle of the study (28 days). The administration of study drug to any patient experiencing DLT in part 1 or part 2 during the first 28 days of treatment was permanently discontinued.
DLT is toxicity meeting any of the following criteria:
any death not explicitly due to underlying disease or external cause
Indication of potential drug-induced liver injury (hyrule cases) as follows:
o ALT or AST >3x upper normal limit (ULN), confirmed by repeated testing after 24 hours, and
o serum Total Bilirubin (TBL) >2 XULN, confirmed by repeated testing after 24 hours
The elevation of otbl and/or AT has no other explanation, such as viral hepatitis (type a, type b or type c), alcoholic or autoimmune hepatitis, existing or acute liver disease, gallbladder obstruction or biliary tract disease, gilbert syndrome, disease progression or other medications that can cause the observed effects.
All grade 4 non-hematologic and hematologic toxicities of any duration
All grade 3 non-hematologic and hematologic toxicities. The exceptions are as follows:
grade o 3 nausea, vomiting and diarrhea, do not require hospitalization or total parenteral nutrition support, and can be managed to grade 2 or less within 48 hours by supportive care.
The o 3 grade electrolyte abnormality was corrected to be less than or equal to 2 grade within 24 hours.
The o 3 grade electrolyte abnormality, lasting <24-72 hours, is clinically uncomplicated and spontaneously regresses or responds to routine medical intervention.
No. 3 grade amylase or lipase, and is irrelevant to symptoms or clinical manifestations of pancreatitis.
End of study definition
The end of the study in part 1 of the study was defined as the point in time at which RP2D had been identified and all patients had been treated with G9.2-17 (IGG 4) until disease progression was confirmed.
The end of study part 2 was defined for each of the three tumor types after completion of Simon two-stage optimization design, and all patients in the group had been treated with G9.2-17 (IGG 4) (alone or in combination) until disease progression was confirmed.
In parts 1 and 2, if patients discontinue treatment for reasons other than disease progression and they do not receive additional systemic anti-cancer therapy, they will receive up to 2 years of OS follow-up after the last dose of G9.2-17 (IgG 4).
The end of the study is defined as the date of the last visit by the last patient.
Test stopping rule
Part 1
If the lower limit of the two items of CI, agrest and Coull, at the lowest study dose level exceeds the target DLT rate, the trial is stopped for safety reasons (Agrest and Coull, 1998).
Part 2
After testing study drugs for 23 patients in phase I of Simon optimization design for CRC and CCA monotherapy groups, if 1 or less patients respond, the corresponding test group is stopped. If the trial proceeds to stage II of Simon's optimization design, the trial group is stopped if the total number of responsive patients in the group is less than or equal to 5.
Simon optimization also guided the test to stop for PDAC G9.2-17 (IgG 4) +gemcitabine/nab-paclitaxel combination treatment group. After the combined G9.2-17 (IgG 4)/chemotherapy test on 11 patients of stage I, the test group was stopped if 6 or less patients exhibited PFS for 6 or more months. If the trial continues to stage II, the trial group is stopped if PFS. Gtoreq.6 months of responding patients total is 16.
To ensure patient safety for both combination treatment groups, a safety break-in was performed, with 8 patients in front. For each cancer type (e.g., CCA, CRC, and/or PDAC), the group was continued only if less than or equal to 2 patients developed DLT (below 25% Target Toxicity Level (TTL)). If 3 or more patients with a given cancer type develop DLT in the combination treatment group, the cancer type is terminated into the group.
Study population
Inclusion criteria
Participants were eligible for inclusion in the study only if all of the following criteria were applicable:
part 1 and part 2
1. Written informed consent (patients with normal spirit, able to understand and willing to sign informed consent)
2. Male or non-pregnant female with age equal to or greater than 18 years old
3. Histologically confirmed unresectable metastatic cancers (allowing for adenocarcinoma and squamous cell carcinoma). Patients with resectable disease are excluded.
4. Can follow the research scheme
5. Life expectancy >3 months
6. Eastern tumor cooperative group (ECOG) expression status 0-1
7. Coronavirus SARS-CoV-2 (COVID-19) negative patient
8. Patients can and are willing to receive pre-treatment and mid-treatment/post-treatment biopsies. Planned biopsies should not expose the patient to a significantly increased risk of complications. All efforts are made to biopsy the same lesion in repeated biopsies.
9. Disease measurable according to the solid tumor Response Evaluation Criteria (RECIST) v 1.1. Note that lesions intended for biopsy should not be target lesions.
10. Sufficient hematology and end organ function is defined by the following laboratory results obtained prior to the first dose of study drug treatment:
a. neutrophil count is greater than or equal to 1x10 9 /L
b. Platelet count is greater than or equal to 100x10 9 L; for hepatocellular carcinoma (HCC) in part 1, > 50X10 9 /L
c. Hemoglobin not transfused in the previous week is more than or equal to 9.0g/dL
d. Creatinine is less than or equal to 1.5x Upper Limit of Normal (ULN)
e. Aspartate aminotransferase AST (SGOT). Ltoreq.3XULN (5 XULN when HCC or liver metastasis is present)
f. Alanine aminotransferase (ALT [ SGPT ]) 3x ULN (5 x ULN when HCC or liver metastasis is present)
g. Bilirubin is less than or equal to 1.5 ULN (patients known to have Gilbert's disease may have bilirubin less than or equal to 3.0 ULN)
h. Albumin not less than 3.0g/dL
i. International Normalized Ratio (INR) and Partial Thromboplastin Time (PTT) less than or equal to 1.5 XULN
j. Amylase and lipase are less than or equal to 1.5 XULN
11. There was no evidence of active infection or infection requiring parenteral antibiotics, and no severe infection within 4 weeks prior to study initiation
12. Women with fertility must be negative for pregnancy tests within 72 hours before treatment begins. For women with fertility: consent was given to maintain abstinence (avoiding idiosyncratic exchange) or to use contraceptive methods with a annual failure rate of <1% during the treatment period and for at least 180 days after the last study treatment.
A woman is fertility if the woman is in a post-menopausal state (continuous amenorrhea. Gtoreq.12 months and no clear cause other than menopause) after the beginner and has not received surgical sterilization (ovariectomy and/or uterus).
Examples of contraceptive methods with a annual failure rate of <1% include bilateral tubal ligation, male sterilization, hormonal contraceptives to inhibit ovulation, hormonal-releasing intrauterine devices and intrauterine copper devices. The reliability of sexual abstinence should be assessed according to the duration of the clinical trial, the patient's preference and the usual lifestyle. Periodic abstinence (e.g., calendar, ovulation, symptomic-thermal) or post-ovulation methods) and in vitro ejaculation (withdrawal) are unacceptable contraceptive methods. Fertility men must employ effective contraceptive methods during the study unless there is a record of infertility.
13. From the last dose of anti-cancer therapy prior to the first administration of G9.2-17 (IgG 4), there is four (4) weeks or 5 half-lives (whichever is shorter)
14. Allowing continued use of bisphosphonate treatment (e.g., zoledronic acid) or Deshumab to treat bone metastases that have been stabilized for at least 6 months prior to C1D1
15. Allowing biliary or gastric outlet obstruction provided that effective drainage is achieved by endoscopic, surgical or interventional means
16. Allowing pancreatic, biliary or intestinal fistulae, provided that they are controlled with appropriate uninfected and unobstructed drainage tubes (if any are in place, it is necessary to confirm patency before the study begins)
In addition, for part 1 only:
17. patient:
a. has received at least one prior line number of systemic therapies for treating metastatic disease, or
b. With tumor types for which no standard care selection is available.
In addition, for part 2 only:
pdac extension group: 1-line metastatic patients who were first receiving a gemcitabine-containing regimen or who were previously untreated with a gemcitabine-containing regimen in a neoadjuvant or adjuvant/locally advanced setting for at least 3 months
CRC and CCA extended group-patients who have received at least one previous line number of therapy in a metastatic environment
Exclusion criteria
Participants were excluded from the study if any of the following criteria were applicable:
1. patients who are reluctant or unable to follow the regimen
2. Patients diagnosed with primary unknown metastatic cancer
3. Previously or now illicit drug addiction (medical and recreational cannabis/Cannabidiol (CBD)/Tetrahydrocannabinol (THC) is not considered "illicit")
4. Clinically significant, uncontrolled bleeding, and any patient with bleeding physique (e.g., active peptic ulcer disease). Allowing prophylactic or therapeutic use of anticoagulants.
5. Pregnant and/or lactating women
6. Any other study agent or any other clinical trial involving another study agent for treatment of solid tumors was received 4 weeks prior to study cycle 1, day 1, or within 5 half-lives of the administered drugs (whichever was shorter), or other study therapy or major surgery was received within 4 weeks of the consent date, or surgery was planned within 4 weeks of the start of the envisaged study (including dental surgery).
7. Radiation therapy was received within 4 weeks of the first dose of study drug, except for limited field palliative radiation therapy, such as used to treat bone pain or focal pain tumor mass, and without compromising the measurable lesions required for response assessment (RECIST v 1.1).
8. Patients with fungal tumor masses
9. Patients with locally advanced PDAC without distant organ metastatic deposition
10. Grade 4 immune-mediated toxicity was generated with previous checkpoint inhibitors. Grade 2 or grade 3 pneumonia or any other grade 3 checkpoint inhibitor-associated toxicity leading to discontinuation of immunotherapy treatment. Low levels (< 3 levels) of toxicity are allowed, such as neuropathy, manageable electrolyte abnormalities and lymphopenia, hair loss and vitiligo resulting from previous treatments.
11. With a history of a second malignancy, those previously treated healthily for more than five years without recurrence or with a low likelihood of recurrence (e.g., non-melanoma skin cancer, cervical cancer in situ, early (or localized) prostate cancer, or superficial bladder cancer)
12. Active brain or pia metastasis. Brain transfer patients are eligible, provided they show clinically and radiologically Stable Disease (SD) at least 4 weeks after definitive therapy, and no steroid (> 10 mg/day of prednisone or equivalent) is used at least 4 weeks before the first dose of study drug.
13. There is evidence of severe or uncontrolled systemic disease, congestive heart failure > New York Heart Association (NYHA) class 2, myocardial Infarction (MI) within 6 months, or laboratory findings that the patient is not suitable for participation in the trial
14. Any medical condition that is considered to seriously jeopardize the patient's safety or that affects the interpretation of the G9.2-17 (IgG 4) toxicity assessment
15. Severe non-healing wounds, active ulcers or untreated fractures
16. Uncontrolled pleural effusion, pericardial effusion or ascites, require repeated drainage procedures. For the purposes of this study, "recurrence" was defined as ≡3 drainage times over the first 30 days.
17. Has a history of severe allergic, allergic or other hypersensitivity reactions to chimeric or humanized antibodies or fusion proteins
18. Significant vascular disease (e.g., aortic aneurysm requiring surgical repair or recent arterial thrombosis) occurs within 6 months of cycle 1 day 1
19. A history of pulmonary embolism, stroke or transient ischemic attack within 3 months prior to cycle 1, day 1
20. History of abdominal fistulae or gastrointestinal perforation 6 months prior to cycle 1 day 1
21. Active autoimmune disorders (type I/II diabetes mellitus, hypothyroidism requiring only hormone replacement, vitiligo, psoriasis or alopecia areata excluded)
22. Systemic immunosuppression therapies are needed including, but not limited to, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. Patients who have received or are receiving acute, low doses of systemic immunosuppressant drugs (e.g.,. Ltoreq.10 mg/day of prednisone or equivalent) may be included in the group. Replacement therapy (e.g., thyroxine, insulin, physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency [ e.g.,. Ltoreq.10 mg/day prednisone equivalent ]) is not considered a form of systemic treatment. The use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular and ophthalmic steroids is allowed.
23. Severe tumor-associated pain (grade
24. Hypercalcemia (according to CTCAE v 5.0,3 grade), although bisphosphonates are used
25. Any other disease, metabolic dysfunction, physical examination findings or clinical laboratory findings, such that a reasonable suspicion of diseases or conditions that prohibit the use of research drugs or possibly affect the interpretation of the results or put the patient at high risk of treatment complications
26. Receiving organ transplants
27. Patients undergoing dialysis
28. For part 1, patients with metastatic castration-resistant prostate cancer are allowed to continue hormone androgen deprivation therapy.
29. Any ablative therapy (radiofrequency ablation or percutaneous ethanol injection) was performed on HCC <6 weeks prior to entry into the trial
30. Hepatic encephalopathy or severe liver adenoma
child-Pugh score of 7 or more
In addition, for part 2 only:
32. hypersensitivity to active substance or any excipient of gemcitabine/nab-paclitaxel
Study of drugs and other interventions
Research interventions are defined as any research agent, market product, placebo or medical instrument intended to be administered/used to study participants according to a research regimen.
Pharmaceutical agent administered in combination with G9.2-17 (IgG 4)
Gemcitabine plus nab-paclitaxel
Gemcitabine is a nucleoside metabolic inhibitor suitable for use alone or in combination with other therapies in the treatment of multiple cancer types. nab-TaxolProtein knotPaclitaxel in a synthetic form) is a microtubule inhibitor and is also useful in the treatment of a variety of tumor types. Specifically, nab-paclitaxel in combination with gemcitabine is used as a first line treatment for metastatic PDAC. Gemcitabine was administered at 1000mg/m2 doses over 30 minutes on days 1, 8 and 15 of each 28 day cycle. Nab-paclitaxel was administered at doses of 125mg/m2 over 3-40 minutes on days 1, 8 and 15 of each 28 day cycle.
Adverse events due to the gemcitabine+nab-paclitaxel combination are shown in table 8 below.
TABLE 8 adverse events reported for gemcitabine + Nab-paclitaxel treatment of pancreatic cancer (based on frequency)
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Limiting the infusion of nab-paclitaxel to 30 minutes reduces the likelihood of infusion-related reactions.
Pre-administration of nab-paclitaxel is not typically required to prevent allergic reactions prior to administration. Gemcitabine is disabled in patients allergic to gemcitabine.
Abraxane causes bone marrow suppression and when neutrophils count<1,500 cells/mm 3 Should not be used at all. Abraxane is known to undergo severe hypersensitivity reactions. Patients known to be sensitive to gemcitabine should not be administered gemcitabine.
The recommended dose modifications of gemcitabine + Nab-paclitaxel in PDAC patients are provided in tables 9-11 below.
TABLE 9 dose level reduction of gemcitabine and nab-paclitaxel
Dosage level Nab-paclitaxel (mg/m) 2 ) Gemcitabine (mg/m) 2 )
Full dose 125 1000
Dose reduction 1 st time 100 800
Dose reduction 2 nd time 75 600
If additional dose reduction is required Suspension of Suspension of
TABLE 10 dose advice and modification for neutropenia and/or thrombocytopenia at the beginning of the cycle or during the cycle
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TABLE 11 dose modification for other adverse reactions
* Toxicity was graded according to NCI CTCAE V.
Study of intervention administration
All patients received G9.2-17 (IgG 4). G9.2-17 (IgG 4) was administered weekly or every 2 weeks via IV infusion until disease progression, unacceptable toxicity occurred, or consent was withdrawn.
In section 1, patients received G9.2-17 (IgG 4) alone, with doses increasing sequentially starting at 0.2 mg/kg.
In section 2, the patient received either G9.2-17 (IgG 4) of RP2D as a single agent (as determined in section 1) or G9.2-17 (IgG 4) RP2D-1 in combination with gemcitabine/nab-paclitaxel as follows:
CRC or CCA patient
o in CRC, G9.2-17 (IgG 4)
o in CCA, G9.2-17 (IgG 4)
PDAC patient
o G9.2.2-17 (IgG 4) +Gemcitabine/nab-Taxol
Other solid tumor types (based on data from section 1)
o G9.2.2-17 (IgG 4), as a single agent and/or in combination with a checkpoint inhibitor or chemotherapy, based on each tumor type
A summary description of each study intervention is understood with reference to table 12.
Patients experiencing DLT in part 1 did not resume treatment. Patients experiencing DLT in part 2 discontinue treatment. If they are experiencing clinical benefit, their treatment may be resumed with the same or reduced dose of G9.2-17 (IgG 4).
TABLE 12 summary features of study interventions
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Preparation of G9.2-17 (IgG 4)
The manufacture and packaging of research pharmaceutical (IMP) G9.2-17 (IgG 4) meets applicable current good manufacturing practice (Good Manufacturing Practice, cGMP) and the product meets standards applicable to the human body.
The G9.2-17 (IgG 4) drug was diluted to the target dose prior to administration. All dilutions should be made in a controlled and sterile environment (patient doses prepared and delivered via about 60 minutes IV infusion).
G9.2-17 (IgG 4) is a sterile liquid and is stored at 2℃to 8℃and protected from light.
Dose decrementing
The G9.2-17 (IgG 4) dose was sustained for patients in the G9.2-17 (IgG 4) +gemcitabine/nab-paclitaxel group
If the patient is experiencing clinical benefit and regimen efficacy assessment criteria from G9.2-17 (IgG 4) and the patient is experiencing adverse reactions not attributable to G9.2-17 (IgG 4), treatment with G9.2-17 (IgG 4) alone may be continued while gemcitabine/nab-paclitaxel is modified/discontinued.
G9.2-17 (IgG 4) may continue if:
the clinical status of the patient does not deteriorate rapidly; and
suspending the combination agent due to AE of the combination agent alone.
If IMAR onset/recurrence cannot be managed by reducing the dose of either agent, both study medications must be discontinued.
Albumin-bound paclitaxel is not recommended in patients with total bilirubin >5x ULN or AST >10x ULN. In addition, the use of albumin-bound paclitaxel is not recommended in metastatic pancreatic adenocarcinoma patients with moderate to severe liver injury (total bilirubin >1.5x ULN and AST +.10x ULN). For patients with moderate or severe liver injury, the initial dose should be reduced.
Discontinuation of research intervention
In rare cases, it may be necessary for the patient to permanently discontinue the study intervention. If study intervention is permanently stopped for reasons other than disease progression and the patient is not treated with other anti-cancer therapies, the patient is continuously assessed for disease progression for up to 2 years. See SoA for data to be collected at the time of discontinuation of study intervention and follow-up and any further evaluation that needs to be completed.
Researchers must take all the effort to keep patients on receiving study treatment until one of the causes of study treatment termination (disease progression, development of toxicity associated with study drugs, withdrawal of consent) is met. If the patient has radiological progress but no clear clinical progress and no replacement therapy has been initiated, the patient may continue to receive study treatment. However, if the patient has clear clinical progress without radiological progress, the study treatment should be stopped and the patient is advised of the available treatment options.
The patient may discontinue before disease progression for any of the following reasons:
DLT occurs according to the definition in section 3.4.4.
AE occurs/recurs outside the DLT window, requiring discontinuation of study treatment
IMAR occurrence/recurrence, need to discontinue study treatment
PureTech Health, LLC termination study
Preventing further administration of the treatment or complications or medical conditions that may compromise patient safety if the study treatment is continued
Pregnancy
Use of non-regimen anti-cancer therapies
Patients may also discontinue before disease progression for any of the following reasons:
patient partial significant deviation regimen (including lack of compliance)
The cause of discontinuation of study treatment should be recorded in the Case Report Form (CRF). If the patient discontinued study treatment due to toxicity, a "dose limiting toxicity" or "adverse event" was recorded as the primary cause of withdrawal. If the patient ceases the study at any time earlier due to AE or SAE, the patient must be followed until they resolve to grade 2 or lower unless improvement is unlikely due to underlying disease.
Concomitant therapy
Any drugs or vaccines (including over-the-counter or prescription drugs, recreational drugs, vitamins and/or herbal supplements) that the participants are receiving at the time of enrollment or during the study must be recorded along with the following information:
cause of use
Date of application, including start and end date
Dose information, including dose and frequency
Granting medication
The following concomitant medications are allowed:
any standard of care pre-operative medication of patients receiving the combination treatment regimen.
Continuing treatment with bisphosphonate therapy (e.g., zoledronic acid) or Deshumab to treat bone metastasis that has been stabilized for at least 6 months prior to treatment (C1D 1)
Use of inhaled corticosteroids and mineralocorticoids (e.g., fludrocortisone), topical steroids, intranasal steroids, intra-articular and ophthalmic steroids
Prophylactic or therapeutic use of anticoagulants
Vaccines that allow for vaccination with covd-19, common influenza and/or other common clinically desirable indications (e.g., tetanus, pneumococci, HBV, etc.) prior to or during the study period. The time and type of vaccination must be recorded.
Illicit drugs
The following drugs were not allowed to be used in this study:
for any indication, other study agents than G9.2-17 (IGG 4) were administered simultaneously.
Systemic immunosuppressive therapy including, but not limited to, cyclophosphamide, azathioprine, methotrexate, thalidomide, and anti-TNF agents. However, patients are allowed to take acute, low dose systemic immunosuppressant medications (e.g., 10mg day's prednisone or equivalent).
Replacement therapy (e.g., thyroxine, insulin, physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency [ e.g.,. Ltoreq.10 mg/day of prednisone equivalent ]) is not considered a systemic treatment modality.
Supportive care
Patients should receive full supportive care including blood transfusions and blood products, as well as treatment with antibiotics, antiemetics, antidiarrheals and analgesics, and other care deemed appropriate and in compliance with institutional guidelines during the study
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Study evaluation and procedure
A signed written ICF approved by the Institutional Review Board (IRB) must be obtained from a potential patient before he/she can participate in any particular study procedure, including a particular study screening procedure.
Once all screening procedures are completed and the patient is determined to meet all qualification criteria, the patient may enter the study.
The study procedures of part 1 groups 1-6 and their corresponding schedules are summarized in SoA (Table 13). The study procedures of part 1 groups 7 and 8 and their corresponding schedules are summarized in SoA (table 14) and do not allow protocol exemption or exemption.
Compliance with all study requirements (including those specified in SoA) is critical and essential for study performance.
When an emergency safety issue occurs or is appreciated, a discussion should be immediately made to determine if intervention is required or to abort the study.
All screening evaluations must be completed and reviewed to confirm compliance of potential participants with all qualification criteria. A screening log is maintained to record details of all participants being screened and to confirm qualifications or record reasons for screening failure (if applicable).
Procedures performed as part of the participants routine clinical management (e.g., blood count) and obtained prior to signing the ICF can be used for screening or baseline purposes provided that the procedures meet the criteria specified by the protocol and are performed within the timeframe defined in the SoA.
Assessment by visit
The SoA (tables 13 and 14) provides a list of evaluations performed during the screening period (up to 28 days), the treatment period (presented as a 28 day cycle), the treatment end/premature termination period, IMAR follow-up and long term follow-up periods. If a medical indication is present, an optional visit is allowed during each treatment cycle during which any study evaluation may be performed.
During the popularity of covd-19, many governments require citizens to maintain social distance and suggest more vulnerable groups to self-quarantine. These types of limitations may affect the ability to conduct this clinical study as originally planned. Planned study center visits may be adjusted so that the study may continue safely during the pandemic. Possible modifications may include:
Visit and/or study program delay
Replacement with a phone/video call
Replacement with home visit
Visit to an alternative clinical site
Performing the visit by a healthcare provider outside the research team
Visit and/or study procedure is completely cancelled.
Screening period (between day-28 and day-1)
The following procedure must be performed within 4 weeks after the initiation of treatment:
study procedure and examination
Written informed consent
Inclusion and exclusion criteria to verify patient eligibility
Patient demographics
Medical history
Previous and concomitant medications
ECHO/Multi-Gate acquisition scan (MUGA)
12 lead ECG (QT interval corrected using the Fridericia formula [ QTcF ])
Physical examination-neurological examination in patients with stable and pre-treated brain metastases
ECOG performance status
Vital signs
Tumor imaging assessment (computed tomography [ CT ] or Magnetic Resonance Imaging (MRI) with or without contrast agent; or Positron Emission Tomography (PET) -CT; CT with contrast agent is preferred)
Clinical laboratory
Pregnancy test, suitable for women with fertility (WOCBP)
Hematology (Hematology)
Serum chemistry
Thyroid Stimulating Hormone (TSH), free T4 or thyroxine (fT 4), serum lipase, amylase, parathyroid hormone (PTH), follicle Stimulating Hormone (FSH), luteinizing Hormone (LH), free cortisol
Coagulation of blood
Urine analysis
Pharmacodynamics and pharmacokinetics
Tumor biopsy
If a biopsy is considered to be at risk to the patient, this procedure may be omitted.
If biopsies are not available, the research center should make all efforts to obtain archived tumor tissue samples that are available as formalin-fixed paraffin-embedded (FFPE) blocks. Acceptable archived samples include those obtained through core needle biopsy or resection procedures over the past five years.
dMMR-MSI-H status (if the MMR and MSI status of the patient were not previously determined, then a check must be made at the local laboratory)
Tumor type-related biomarkers
Treatment period
Each treatment cycle had a duration of 28 days.
Treatment procedure on day 1 of each cycle (CXD 1; day 2 from cycle 2)The following procedure was performed on day 1 of each treatment cycle.
Study procedure and examination
Concomitant medication
·AE
12 lead ECG (QTcF)
Physical examination
ECOG performance status
Vital signs
Clinical laboratory
WOCBP pregnancy test
Hematology (Hematology)
Serum chemistry
TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol
Coagulation of blood
Urine analysis
PK/PD assessment
PD blood sampling
PK blood sampling
ADA blood sampling
Tumor type-related biomarkers
Study drug administration
Administration was only after all pre-dose evaluations and procedures were completed.
In addition, from day 1 of cycle 3, the following evaluations were performed every 8 weeks:
tumor imaging assessment (CT or MRI with or without contrast agent; or PET-CT; CT with contrast agent being preferred)
Furthermore, starting on day 1 of cycle 4, the following evaluations were performed every 3 months:
·ECHO/MUGA
groups 1-6: treatment course on days 2 and 8 (CXD 2+ -1 and CXD 8+ -1) of cycle 1 and 3 Sequence of steps
Study procedure and examination
Concomitant medication
·AE
PK/PD assessment
PD blood sampling
PK blood sampling
Groups 1-6: day 15 of each cycle (cycle 1 is CXD15+ -1 day and starting from cycle 2 is+ -2) Day) treatment program
The following procedure was performed on day 15 of each treatment cycle.
Study procedure and examination
Concomitant medication
·AE
Physical examination
ECOG performance status
Vital signs
Clinical laboratory
Hematology (Hematology)
Serum chemistry
Coagulation of blood
Urine analysis
PK/PD assessment
PD blood sampling at C1D15 and C3D15 only
PK blood sampling at C1D15 and C3D15 only
Tumor type-related biomarkers
Tumor biopsies were taken on C3D 15.+ -. 7 days (only on cycle 3; if the risk was considered too great for the patient, it could be cancelled)
Study drug administration
Administration was only after all pre-dose evaluations and procedures were completed.
Groups 7 and 8: treatment program on cycle 1 and 3 day 3 (C1D 3.+ -. 1 day and C3D 3.+ -. 1 day)
Study procedure and examination
Concomitant medication
·AE
PK/PD assessment
PD blood sampling
PK blood sampling
Groups 7 and 8: treatment program on day 8 of each cycle (CXD 8.+ -. 1 day)
Study procedure and examination
Concomitant medication
·AE
Physical examination
ECOG performance status
Vital signs
Clinical laboratory
Hematology (Hematology)
Serum chemistry
Coagulation of blood
Urine analysis
PK/PD assessment
PD blood sampling
PK blood sampling only in odd cycles
Study drug administration
Administration was only after all pre-dose evaluations and procedures were completed.
Groups 7 and 8: 1 st of each cycleDay 5 and day 22 (cycle 1 is CXD 15.+ -. 1 day and start from cycle 2) Beginning with ±2 days) of treatment procedure
The following procedure was performed on days 15 and 22 of each treatment cycle.
Study procedure and examination
Concomitant medication
·AE
Physical examination
ECOG performance status
Vital signs
Clinical laboratory
Hematology (Hematology)
Serum chemistry
Coagulation of blood
Urine analysis
PK/PD assessment
PD blood sampling at C1D15 and C3D15 only
PK blood sampling only in odd cycles
Tumor type-related biomarkers
Tumor biopsies were taken on C3D 15.+ -. 7 days (only on cycle 3; if the risk was considered too great for the patient, it could be cancelled)
ADA blood sampling at C1D15 and C2D15 only
Study drug administration
Administration was only after all pre-dose evaluations and procedures were completed.
Additional treatment after cycle 4
As indicated in the SoA (tables 13 and 14), the treatment cycle following cycle 4 may be repeated. If the patient is experiencing clinical benefit, the patient can continue treatment even with radiological progress.
Treatment end or early termination procedure
The following procedure, including patients who discontinued treatment prematurely, was performed 30 days (+ -3 days) after the last dose.
Study procedure and examination
Concomitant medication
·AE
Physical examination
·ECOG
Vital signs
Tumor imaging assessment: if the study ended >8 weeks after the previous scan, a confirmatory scan was performed.
Clinical laboratory
WOCBP pregnancy test
Hematology (Hematology)
Serum chemistry
TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol
Coagulation of blood
Urine analysis
PD assessment
PD blood sampling
ADA blood sampling
Tumor type-related biomarkers
Long-term follow-up
OS was assessed every 3 months for up to 2 years after the end/premature termination of patient treatment. For patients who have discontinued treatment for reasons other than disease progression and who have not received additional systemic anti-cancer treatment, tumor imaging assessment should be continued as much as possible.
Survival data is collected at least once every 3 months and information about any new anti-cancer therapies that begin after disease progression. Survival data may be collected more frequently to support data cleansing or regulatory submission work. The follow-up may be through telephone interviews, electronic messages or chart reviews and reported on the CRF. During the follow-up period, the event is collected and reported within 24 hours after the death (regardless of causality) is discovered or notified.
RECIST v1.1 criteria for tumor assessment
In screening tumor evaluation, tumor lesions/lymph nodes were classified as measurable or non-measurable, with measurable tumor lesions (except pathological lymph nodes, which were measured on the shortest axis) recorded according to the longest diameter in the measurement plane. When there is more than one measurable lesion at the time of screening, all lesions representing up to a total of five lesions (and up to two lesions per organ) of all affected organs should be identified as target lesions. The target lesion (the lesion with the longest diameter) should be selected based on its size. The sum of the diameters of all target lesions was calculated and reported as the baseline total diameter.
All other lesions (or disease sites) including pathological lymph nodes should be identified as non-target lesions and should also be recorded at the time of screening. No measurement is required and these lesions should be tracked as "present", "absent" or "clear progression".
According to RECIST v1.1 guidelines (Eisenhauer et al 2009), tumor target lesions were evaluated using the following disease response metrics:
evaluation of target lesions:
complete Response (CR): all target lesions disappeared. The minor axis of any pathological lymph node (whether targeted or non-targeted) must be reduced to <10mm.
Partial Response (PR): the sum of the diameters of the target lesions is reduced by at least 30% with reference to the baseline total diameter.
Progressive disease: the sum of diameters of target lesions increases by at least 20% with reference to the minimum sum at study (which includes the baseline sum if at study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (note: the appearance of one or more new lesions is also considered to be progression).
Stable Disease (SD): taking the minimum overall diameter at the time of investigation as a reference, neither sufficient shrinkage nor sufficient increase satisfies the conditions of PR.
Evaluation of non-target lesions:
CR: all non-lesions disappeared and tumor marker levels were normalized. All lymph node sizes must be non-pathological (short axis <10 mm).
non-CR/non-progressive disease (non-PD): the persistence of one or more non-target lesions and/or tumor marker levels are maintained above normal limits.
Progressive disease: clear progress in existing non-target lesions. (note: the appearance of one or more new lesions is also considered to be progression).
The summary is provided in table 15 below.
TABLE 15 evaluation of overall time point response of patients with measurable disease at baseline
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CR: full response, non-PD: non-progressive disease, PR: partial response, SD: stabilizing disease, NE: not being able to evaluate
* When the target lesions show SD/PR but not some subset of target lesions are not evaluable, care must be taken to decide whether to refer to the overall response at that time point as SD/PR or NE. This is based on whether non-evaluable lesions (if they show growth) can elicit an overall response of progressive disease in the context of other lesion responses seen. If the non-evaluable non-target lesions account for a significant proportion of the overall disease burden, then the appropriate time point response is NE.
The disease response measures at different time points allow the following to be calculated:
disease Control Rate (DCR), defined as the percentage of patients who achieved CR, PR and SD.
Objective Response Rate (ORR), defined as the proportion of patients whose tumor size decreased by a predetermined amount (tumor shrinkage ≡30%).
Progression Free Survival (PFS), defined as the time from the onset of study drug treatment to disease progression (tumor growth. Gtoreq.30%).
Response duration (DoR), defined as the length of time that a tumor continues to respond to treatment without cancer growth or spread.
Total survival (OS), defined as the time from the start of study drug treatment to death due to any cause.
Security assessment
Physical examination
Medical science and physical examination must be performed by qualified physicians, nurse practitioner or physician assistants, and should include thorough examination of all body systems. In addition, height (only at screening) and weight were measured.
Vital signs
Vital signs including body temperature, blood pressure (systolic and diastolic), heart rate and respiration rate were measured in the supine position after 5 minutes of rest.
Electrocardiogram
A 12-lead ECG is obtained as outlined in SoA (see tables 13 and 14) using an ECG machine that automatically calculates heart rate and measures heart rate, PR interval, QRS duration, time distance on the ECG trace from the start of the QRS complex to the end of the T-wave (QT) interval, and QTcF interval.
Clinical safety laboratory assessment
Blood samples from patients were collected according to SoA (tables 13 and 14) for routine clinical laboratory testing (approximately 5mL per time point); additional tests may be performed at any time during the study, if necessary.
Clinical laboratory parameters were analyzed at the local laboratory of the study center. The laboratory assessment completed included hematology and serum chemistry and was defined as follows:
serum chemistry: including glucose, total protein, albumin, electrolytes [ sodium, potassium, chlorine, magnesium, phosphorus ], calcium, bilirubin (total, direct), SGPT (ALT) or SGOT (AST), alkaline phosphatase, gamma glutamyl transferase (gamma GT), lactate Dehydrogenase (LDH), creatinine, hemoglobin A1c (HgbA 1 c) (only in the presence of a history of type 1 or type 2 diabetes), blood urea nitrogen, creatine Phosphokinase (CPK)
o additionally TSH, fT4, lipase, amylase, PTH, FSH, LH, free cortisol at the indicated visit
o assessing fasting blood glucose only in the presence of clinical indications
Hematology: comprises differential whole blood count, platelets, and hemoglobin
Coagulation: including Prothrombin Time (PT) and PTT, activated Partial Thromboplastin Time (APTT) and INR (if anticoagulants are allowed to be used), C-reactive protein (CRP) and troponin
Urine analysis: a urine sample from the patient is collected for routine urine analysis. Urine analysis includes color, appearance and specific gravity paper, proteins, leukocyte esterases, glucose, ketones, urobilinogens, nitrites, white blood cell count (WBC), red blood cell count (RBC) and pH, and urine culture if the patient has clinical symptoms.
If the clinically significant value does not revert to normal/baseline or grade 1 within a reasonable period of time, the etiology should be determined.
Laboratory tests required for all protocols must be performed according to the laboratory manual and SoA (tables 13 and 14).
If laboratory values of non-protocol specified laboratory tests performed at the local laboratory of the institution require changes managed by the participants or are considered clinically significant (e.g., SAE or AE or dose modification), the results must be recorded.
Pregnancy test
WOCBP should be included only after menstrual periods are confirmed and the hypersensitive urine or serum pregnancy test is negative.
Additional pregnancy tests should be performed during the treatment period and at the end of treatment/early termination of visit according to SoA (tables 13 and 14) and according to local requirements.
Pregnancy tests are performed whenever the menstrual cycle is missed or pregnancy is suspected.
If the patient had a history of bilateral tubal ovariectomy and/or hysterectomy, these surgeries are recorded; these patients do not need to be subjected to pregnancy tests.
Pharmacokinetic assessment
The following serum PK parameters for G9.2-17 (IGG 4) were calculated, if possible:
·AUC 0-336h
·C max
·T max
·t 1/2
serum concentration versus time curve
Approximately 5mL of blood samples were collected and processed into serum at each time point as specified in SoA (tables 13 and 14).
PK schedule for groups 1-6:
cycle 1 and 3 day 1
Pre-administration of drug
End of infusion (EOI)
2 hours after EOI (+ -30 minutes)
4 hours after EOI (+ -30 minutes)
Cycle 1 and 3 day 15
Pre-administration of drug
At EOI time
Cycle 1 and 3 days 2 and 8 (day of non-administration)
Any point in time during the visit
Cycle 2 and cycle 4 day 1
Pre-administration of drug
At EOI time
Every 2 cycles and 1 day after the 4 th cycle (i.e. C6D1, C8D1, etc.)
Pre-administration of drug
At EOI time
PK schedule for groups 7 and 8:
day 1 of each odd numbered cycle (i.e., C1D1, C3D1, etc.)
Pre-administration of drug
End of infusion (EOI)
1 hour after EOI (+ -15 minutes)
Day 3 of each odd numbered cycle (i.e., C1D3, C3D3, etc.)
Any point in time during the visit
Odd numbered periods of days 8, 15 and 22 (i.e., C1D8, C3D8, etc.)
Pre-administration of drug
At EOI time
Even numbered period day 1 (i.e., C2D1, C4D1, etc.)
Pre-administration of drug
At EOI time
If it is determined that the dose of study drug is to be discontinued, additional PK and safety assessments are collected after resumption of dosing; additional PK evaluations were performed during the interruptible period. If the dose of study drug is reduced, additional PK assessments are collected before administration of the reduced dose (within 2 hours prior to dosing) and 2 to 4 hours after starting the reduced dose of study drug. Additional PK and other blood assessments may be performed if clinical indications exist. Because of the restriction of covd-19, it is not possible to leave the center of the patient above 2 hours post-dose to provide samples only at EOI and 2 hours post-dose.
Instructions for collecting and processing biological samples are provided. The actual date and time of each sample was recorded (24 hours Zhong Shijian).
The designated laboratory uses samples to evaluate serum concentration levels of total G9.2-17 (IGG 4) and free/partially free G9.2-17 (IGG 4). The concentration was determined using a validated assay. A minimum of two 50. Mu.L serum aliquots were required to determine total G9.2-17 (IGG 4) concentration. A minimum of two 100 μl serum aliquots were required to determine the free and partial free G9.2-17 (IGG 4) concentrations, with the remaining serum in the third aliquot. Samples collected for analysis of G9.2-17 (IGG 4) plasma concentrations may also be used to assess safety or efficacy aspects associated with problems occurring during or after the study.
These blood samples were not subjected to genetic analysis. Participant confidentiality is maintained. In the visit to obtain PD, ADA, safety laboratory blood samples for determining G9.2-17 (IGG 4), a sample of sufficient volume may be used.
Genetics of
The study did not evaluate genetics.
Pharmacodynamic biomarkers
The planned time points for biomarker assessment are provided in SoA (tables 13 and 14); after 6 months of treatment, sampling can be reduced to once every 3 cycles.
Collecting biological samples for other biomarker studies is also part of this study. The following samples were required for biomarker studies and were collected from all participants in the study as specified in SoA:
blood samples collected prior to study drug administration (about 15mL prior to administration)
Tumor biopsy (tissue sample)
PD biomarkers (by flow cytometry, ELISA, IHC, or multiplex phenotyping) of samples were tested to evaluate their association with observed clinical responses to G9.2-17 (IGG 4) using validated assays.
The study assessed the following biomarkers:
tumor markers (blood): CA15-3, CA-125, carcinoembryonic antigen (CEA), CA19-9, alpha-fetoprotein, neuronal Specific Enolase (NSE), cytokeratin fragment-21 (CYFRA-21) were evaluated as tumor type as needed prior to each periodic dose administration. After 6 months of treatment, the time was optionally reduced to once every 3 cycles on the same schedule as the tumor imaging evaluation.
PBMC phenotype (blood): for example, CD3, CD4, CD8, CD45RO, fork frame protein P3 (FOXP 3), CD11B, CD, CD15, CD16, CD33, CD68, human Leukocyte Antigen (HLA) DR, CD163, arginase 1, granzyme B, KI, PD-1, PD L1, ubiquitin (PANCK)
Cytokine (blood): for example, interferon gamma (IFNgamma), IL 10, IL 12p70, IL 13, IL 1 beta, IL 2, IL 4, IL 6, IL 8, TNF alpha, MIP-1b, monocyte chemotactic protein 1 (MCP-1), MIP-1a, IL 17a, IL 5, TGF beta
Gal-9 in blood and tumor tissue
PD-L1 (organization)
Mismatch repair status (tissue)
Tumor Mutation Burden (TMB)
Exploratory biomarker changes (if any) are related to safety and response outcomes.
After the last visit of the last patient of the study, the samples can be stored in the selected facility for up to 2 years (or according to local regulations) to be able to further analyze the effect of G9.2-17 (IGG 4) on the pharmacodynamic biomarkers.
Immunogenicity assessment
Blood samples (approximately 3 mL) were collected from all participants according to SoA (tables 13 and 14) and processed into serum. In addition, serum samples should also be collected from patients who either discontinue study intervention or withdraw from the study at the end of treatment/early termination of visit.
Groups 1-6: 1 st cycle to 4 th cycle, 1 st day
Pre-administration of drug
Groups 1-6: every 2 cycles and 1 day after 4 th cycle (i.e., C6D1, C8D1, etc.):
pre-administration of drug
Groups 7 and 8: day 1 of each cycle
Pre-administration of drug
Groups 7 and 8: day 15 of cycle 1 only and day 15 of cycle 2
Pre-administration of drug
A minimum of two serum aliquots of 500 μl each were obtained, with the remaining serum in the third tube. The samples were transported to a laboratory designated for analysis using a validated assay. These samples were tested.
Serum samples were screened for antibodies (ADA) that bind to G9.2-17 (IgG 4) and the confirmation of titers of positive samples was reported. Other assays may be performed to verify the stability of antibodies G9.2-17 (IgG 4) and/or to further characterize the immunogenicity of G9.2-17 (IgG 4).
Detection and characterization of antibodies to G9.2-17 (IgG 4) was performed using validated assay methods. All samples collected for detection of antibodies to study intervention were evaluated for G9.2-17 (IgG 4) serum concentration to enable interpretation of antibody data. Antibodies can be further characterized and/or evaluated for their ability to neutralize the activity of the intervention. After the last visit of the last patient of the study, the samples can be stored in a suitable facility for up to 2 years (or according to local regulations) to be able to further analyze the immune response to G9.2-17 (IgG 4).
Other evaluation
Demographic profile
At screening, patient demographics were collected. These data include age, gender, race, and ethnicity.
Medical history
Medical history includes tumor history, surgery/transplant history, radiotherapy history, and covd 19 history and detection.
Personal medical history including previous treatment/surgery, record of any in situ or past implants, previous and/or currently used medical devices, concomitant medication (name, indication, dose, route, date of start and end, dose modification (if any) and cause), preexisting symptoms and AEs), risky genetic diseases based on family history and the patient's most known complete family history
Record of any dental treatment performed within 12 months of expiration
For patients who have previously resected pancreatic adenocarcinoma, record whether the primary tumor is located in the head, body or tail of the pancreas.
Defecation habit/typical frequency and viscosity
Record any dietary requirements or preferences (e.g., carrying out a particular dietary regimen: intermittent fasting, ketogenic diet, etc.)
Record past and present allergic conditions (allergen, severity)
Past and concomitant use of drugs
The past and concomitant medications, including vaccines and supplemental treatments/supplements, for each patient were recorded at each scheduled visit (tables 13 and 14).
Tumor imaging assessment
Tumor assessment using CT or MRI with or without contrast agent; PET-CT was performed.
CT with contrast agent is the preferred modality (if CT is not feasible or appropriate given the disease location, MRI, PET-CT or other imaging modality is used instead of or in addition to CT scanning). The assessment should include at least chest/abdomen/pelvis and should include other anatomical regions shown based on the patient's tumor type and/or medical history. The imaging scan must be de-identified and archived in raw format as part of the patient study file. Although the type of scan obtained is suitable for the disease, the same method should be used for the duration of the study.
In the study, the evaluation was performed according to SoA (i.e., C3D1, C5D1, C7D1, C9D1, etc.) once every 8 weeks.+ -. 7 days, and at the end of the treatment if no evaluation was performed within the past 4-6 weeks. The evaluation may be performed more frequently if there is a clinical indication. For part 2 only, if an objective response is observed in the scan, a confirmation scan is performed after 4 weeks (+7 days). After the confirmatory scan, the scheduled scan will resume at a frequency of every 8 weeks (+ -7 days) starting from the day of confirmatory scan.
Tumor biopsy
Pre-treatment and in-treatment biopsies were collected. Pre-treatment biopsies were collected during screening. If a pre-treatment biopsy is not available for reasons outlined in inclusion criteria and the patient is admitted to the group study, an archived tumor tissue specimen of the patient is collected from the primary tumor and/or metastatic deposit. Resections or core biopsies (FFPE tissue blocks or fresh tissue in formalin) are obtained currently or within 5 years of the beginning of the study from primary tumor lesions or metastatic deposits. If both primary and metastatic tissue is available, metastatic deposit tissue is preferentially used. If treatment information received before and after tissue acquisition is available, this information is also collected.
In-treatment biopsies are scheduled at C3D15±7 days and should be performed only after the tumor imaging scan of cycle 3. In the event that surgery cannot be performed within the time frame prescribed by the protocol, an alternative may be allowed, but must be discussed with the study master/medical supervisor. It is believed that a variety of clinical factors may lead to difficulty in obtaining a sufficient sample. The decision to not complete the biopsy in treatment should be discussed with the medical inspector.
ECHO/MUGA
ECHO and/or MUGA were obtained at the time points indicated in SoA (tables 13 and 14). If clinical indications are present, the assessment will be repeated every 3 months.
ECOG
ECOG performance status was assessed at the time points indicated in the SoA (tables 13 and 14) using the following ratings (Oken et al, 1982).
Stage 0: is fully active and can perform all pre-disease manifestations without limitation
Stage 1: strenuous physical activity is limited but can be ambulatory and enables mild or sedentary tasks, such as mild household, office tasks
Stage 2: can be ambulatory and self-care, but cannot perform any work activities. Can get up and walk, and the awake time is more than 50 percent
Stage 3: can only carry out limited self-care, is limited on a bed or a chair, and has a waking time of more than 50 percent
Stage 4: completely disabled. No self-care can be performed. Is completely limited on the bed or the chair
Stage 5: death of
Adverse Event (AE), serious Adverse Event (SAE) and other security reports
AE is defined in the ICH GCP guidelines as "any adverse medical event that occurs in a patient administered a pharmaceutical product or in a clinical study patient, and is not necessarily causally related to the treatment.
This definition of AE was extended in this study to include the occurrence of any such event (e.g., sign, symptom or diagnosis) or worsening of a preexisting medical condition from the time the patient signed an informed consent to the time the study medication began to be used. Exacerbations indicate an increase in the severity, frequency, or duration of the condition, or associated with significantly worse results, of a preexisting medical condition (e.g., diabetes, migraine, gout, hypertension, etc.).
Serious adverse events
SAE is defined as the following AE:
leading to death;
life threatening (exposing the patient to risk of direct death);
requiring hospitalization or extending the existing hospitalization time;
hospitalization consistent with the definition of "severe" is the admission of any hospitalized patient, including at least overnight at a healthcare facility. Admission of inpatients does not include: rehabilitation facilities, point-of-care facilities, professional care facilities, nursing homes, conventional emergency room access, surgery on the same day (as outpatient/daytime/non-hospitalization) or social admission (e.g., where the patient is not sleeping).
Resulting in persistent or severe disablement/incapacitation; or (b)
Congenital abnormalities/birth defects
An important medical event that may not lead to death, life threatening or require hospitalization may jeopardize the patient and may require medical or surgical intervention to prevent occurrence of one of the results listed in this definition, if it is determined by appropriate medical judgment, the event may be regarded as SAE. Examples of such medical events include allergic reactions and allergic bronchospasms, blood cachexia or tics that require intensive treatment in an emergency room or home, but do not result in hospitalization.
Correlation of
For all AEs, enough information should be obtained to determine causal relationships of AEs (e.g., study drugs or other diseases). The AE versus study treatment was evaluated as defined below:
irrelevant: any event that does not follow the reasonable temporal order of study drug administration and that may result from the clinical state of the patient or other therapeutic pattern administered to the patient.
Unlikely to correlate: no reasonable time sequence of study drug administration is followed or any event that might result from the clinical state of the patient or other therapeutic pattern administered to the patient.
Possible correlation: any response that follows a reasonable time sequence of study drug administration or follows a known pattern of response to a suspected drug and that cannot be reasonably interpreted by known features of the patient's clinical state or other pattern of therapy administered to the patient.
Correlation: any response that follows a reasonable time sequence of study drug administration and follows a known response pattern to suspected drugs and recurs with re-challenge and/or is ameliorated by stopping the drug or reducing the dose.
Adverse event management
AE was not recorded prior to administration of the study medication of the first dose. The beginning AE or symptoms associated with medical history that worsened after study drug administration were recorded. AE should be tracked until they resolve, return to baseline or are determined to be stable or chronic. All SAE were collected until 30 days after the last dose of study medication. All SAE's associated with the study procedure must be collected from the date of patient written consent.
Immune-mediated adverse reactions
The specific IMAR indicated is:
immune-mediated hepatitis
Immune-mediated nephritis
Immune mediated pneumonia
Immune mediated pneumonia
Immune-mediated colitis and diarrhea immune-mediated endocrinopathy
Immune mediated skin reactions
Other immune-mediated adverse reactions: arthritis, encephalitis, rhabdomyolysis, myositis, myocarditis, pancreatitis, and uveitis.
The monitoring program is intended to limit the severity and duration of IMAR occurring during the development of the combination drug and includes: physical examination, vital signs, safety laboratory assessment (including hematology, biochemistry), endocrine function assessment on each day 1 of the new dosing cycle (pre-dosing), coagulation status assessment and urine analysis were performed at the scheduled visit. The evaluation schedule (tables 13 and 14) also covers evaluating ejection fraction once every three months and performing periodic ECG.
A summary of management of IMAR by G9.2-17 (IgG 4), alone or in combination with other therapeutic agents, is provided in table 16 below.
TABLE 16 management of immune-mediated adverse reactions (IMARs) by G9.2-17 (IgG 4)
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Dose reduction procedure for adverse event management
If dose reduction is used for AE management in part 2 of the study, two dose reductions of 50% each are allowed. Additional dose reduction is performed when clinical benefit is expected and may continue to be obtained.
Clinical laboratory abnormalities and other abnormalities assessment as AE and SAE
If the definition of AE or SAE is met, then abnormal laboratory results (e.g., clinical chemistry, hematology, and urine analysis) or other abnormal assessments (e.g., ECG or vital signs) that are judged clinically significant are recorded as AE and SAE. Clinically significant abnormal laboratory results or other abnormal assessments detected during the study or present at the time of screening and significantly worsening after study initiation were reported as AE or SAE. However, clinically significant abnormal laboratory findings or other abnormal assessments are associated with the disease being studied (unless judged to be more severe than the patient's condition is expected) or are present or detected at the beginning of the study and are not worsening, will not be reported as AE or SAE.
Laboratory measurements that deviate clinically significantly from previous measurements can be repeated. Additional or more frequent checks than prescribed in the protocol should be made if necessary to provide adequate AE recordings and AE resolution.
Time period and frequency for collecting AE and SAE information
All AEs and SAE were collected from the intervention at the time points specified in the SoA (tables 13 and 14) until the follow-up visit.
Medical events that begin before study intervention begins but after informed consent is obtained are recorded as medical history/current medical condition, rather than AE.
All SAE are recorded and reported immediately, and should not exceed 24 hours in any case.
Follow-up for AE and SAE
After the initial AE/SAE report, each participant is required to be actively tracked at the time of subsequent visits/contacts. All SAE are tracked until resolved, stable, event gets other explanation or participants lose access.
Statistical considerations
When the last patient received the last visit, the study was completed. After the last patient had a primary endpoint event, the database was locked for primary analysis. Final study analysis was performed after study completion.
Statistical assumptions
The current study was designed to identify the MTD of G9.2-17 (IgG 4) (part 1) by evaluating DLT, followed by a two-stage optimization design of Simon to evaluate drug activity in three disease types (alone or in combination). The study of section 2 is assumed to be detailed below.
CRC and CCAG9.2-17 (IgG 4) monotherapy groups
Null hypothesis: ORR 3 is less than or equal to 5 percent
Alternative assumptions: ORR 3 is more than or equal to 15 percent.
PDAC G9.2-17 (IgG 4) +gemcitabine/nab-paclitaxel combination therapy
In a first line metastatic environment using gemcitabine/nab-paclitaxel, PFS 6 was reported to be 50% (Von Hoff et al, 2013). The study tested the following:
null hypothesis: PFS 6 is less than or equal to 50 percent
Alternative assumptions: PFS 6 is 75%.
Analysis set
Unless otherwise specified, the intent-to-treat (ITT) population is defined as those patients who receive at least one dose of study medication. The ITT was subjected to primary efficacy analysis. Patient treatment is performed on ITT.
Efficacy populations are defined as all patients in ITT who underwent at least one measurable ORR 3 or PFS 6 assessment. This population was used for sensitivity analysis.
A population meeting regimen (PP) is defined as any patient who receives G9.2-17 (IGG 4) for at least one complete cycle without significant regimen deviation.
A safety group (SAF) is defined as all patients receiving at least one dose of study drug. Security analysis was performed on SAF.
The PK/PD population is defined as those patients who have received G9.2-17 (IGG 4) for at least one complete cycle.
Primary endpoint
Security analysis-part 1 and part 2
All security analyses were performed on SAFs unless otherwise specified.
Adverse events
Adverse Events (TEAEs) occurring during treatment are defined as events that occur at or after the first dose of study medication. The MedDRA coding dictionary is used for the coding of AE. TEAE, severe or CTCAE grade 3 or 4 TEAE and treatment-related TEAE are summarized generally in terms of system organ classification and treatment group preference. These summarize the number of events and the number and percentage of patients for a given event. In addition, the number and percentage of TEAE patients are provided in terms of maximum severity. A summary of all TEAEs (by systemic organ category and preferred terminology) occurring in ≡5% of patients in either treatment group is provided.
DLT, MTD and RP2D are summarized.
Laboratory assessment
All laboratory-based data are presented as a list of all values and abnormal results that are judged clinically significant (which are reported as AEs). The per-visit and treatment groups provided a numerical summary of all observed results and changes assessed relative to the baseline screening laboratory, including chemical, hematology, and urinalysis results. No inference comparisons were planned.
Vital signs
A summary of all observed results and numerical changes in screening vital signs from baseline, including blood pressure, heart rate, respiration rate, and body temperature, are provided by time points and treatment groups. No inference analysis of vital signs is planned.
ECG, ECHO/MUGA and physical examination
The physical examination data and changes are presented as a list. Based on the occurrence of clinically significant abnormalities, the ECG results are presented as a list and summarized by treatment group and visit. No inference comparisons across treatment groups were planned.
Principal efficacy analysis-part 2
Disease response was assessed according to RECIST v1.1 and descriptive summary was made for ITT, PP and efficacy populations.
The main curative effect end point is:
ORR 3 of CRC and CCA
PFS 6 of PDAC
Secondary endpoint
Pharmacokinetics, pharmacodynamics and immunogenicity
PK, PD and immunogenicity were summarized descriptive for PK/PD populations in part 1 and part 2.
Secondary efficacy analysis-part 2
Disease response (ORR, PFS, DCR, doR and OS) was assessed according to RECIST v1.1 and descriptive summary was made for ITT, PP and efficacy populations.
Exploratory endpoint
The analysis of exploratory endpoints is detailed in SAP.
Other analyses
Other collected data not explicitly mentioned are presented in the patient list.
Distribution, demographics, baseline characteristics, and medical history
Treatment information is summarized, including the number of patients in the group, screening failures, the number of patients treated and patients withdrawn for reasons.
Demographic data, baseline characteristics, and medical history were statistically summarized per treatment group and for ITT and PP overall usage descriptions.
Past and concomitant use of drugs
The number and percentage of patients taking past and concomitant medications were summarized in terms of treatment groups and for ITT and PP overall.
Example 3 non-GLP Single dose, range-exploring intravenous toxicity study in Male Sprague Dawley rats with 1 week and 3 week post-dosing observation period
The study evaluated the anatomical endpoints of G9.2-17IgG4 following necropsy at week 1 (end-stage) and week 3 (recovery stage) following a single intravenous bolus administration to Sprague Dawley rats on days 8 and 22. All animals survived to the predetermined necropsy. In post-mortem animals at the end of the study or recovery period, there were no macroscopic findings, organ weight changes, or microscopic findings associated with the test samples.
The purpose of this non-GLP exploratory, single dose, range exploratory, intravenous toxicity study was to identify and characterize acute toxicity of G9.2-17IgG4 following 2 minutes of intravenous bolus administration followed by 1 week (end) and 3 weeks (recovery) of post-dosing observation period in Sprague Dawley rats.
This non-GLP single dose toxicity study was performed in 24 Sprague Dawley male rats to determine the toxicology and potential toxicity of different doses of G9.2-17IgG4 in a single administration. The animals were administered vehicle or 10mg/kg, 30mg/kg or 70mg/kg G9.2-17IgG4 by slow bolus intravenous infusion over at least 2 minutes on day 1 followed by 1 week (end-stage, day 8) or 3 weeks (recovery stage, day 22) after administration. Study endpoints included mortality, clinical observations, weight and food consumption, clinical pathology (hematology, coagulation, clinical chemistry and urine analysis), toxico-kinetic parameters, ADA evaluation and pathology (gross necropsy, organ weight and histopathology). A summary of the experimental design is provided in table 17 below.
TABLE 17 design of experiment
a Euthanasia was performed on 3 animals/sex/group at end-stage necropsy on day 8; the remaining 3 animals/sex/group were euthanized at necropsy at day 22 recovery.
b The vehicle was formulation buffer (20mM Tris,150mM NaCl,pH 8.0 ±0.05).
All surviving animals were necropsied on either day 8 or day 22. Complete necropsy was performed and organ weights were collected. All animals were weighed at the end and recovery stages. The tissues required for microscopic evaluation were trimmed, routinely treated, paraffin embedded and stained with hematoxylin and eosin.
There were no unexpected deaths during this study. All animals survived to end-stage or convalescent necropsy. The noted histological changes are believed to be occasional findings or associated with certain aspects of the experimental procedure rather than administration of the test article. There were no sample-related changes in the incidence, severity, or histological characteristics of these occasional tissue changes. No G9.2-17IgG 4-related findings were noted in clinical observations, body weight, food consumption, clinical pathology or anatomical pathology. In summary, single intravenous administration of 10, 30 and 70mg/kg G9.2-17IgG4 to Sprague Dawley rats was tolerated without adverse findings. Thus, under the conditions of this study, NOEL was 70mg/kg.
Example 4 non-GLP Single dose, range exploration intravenous infusion toxicity study of G9.2-17IgG4 in cynomolgus monkeys with 3 week post-dosing observation period
This non-GLP single dose toxicity study was performed in 8 cynomolgus monkeys to identify and characterize the acute toxicity of G9.2-17IgG4 administered as a single dose at different doses. The animals (1 male [ M ]/1 female [ F ]/group) were administered vehicle or 30mg/kg, 100mg/kg or 200mg/kg G9.2-17IgG4 by 30 min Intravenous (IV) infusion followed by a 3 week post-administration observation period. The study endpoints included: mortality, clinical observations, body weight, and qualitative food consumption; clinical pathology (galectin 9 expression on hematology, coagulation, clinical chemistry, immunophenotyping and leukocyte subpopulations, and cytokine analysis); a toxicological kinetic parameter; serum collection for potential drug-resistant antibody evaluation (ADA); and soluble galectin-9 assays; and pathology (gross necropsy, organ weight and histopathology).
No G9.2-17IgG 4-related findings were noted in clinical observations, body weight, food consumption, clinical pathology (hematology, clinical chemistry, coagulation or cytokine analysis), immunophenotyping, galectin-9 expression on leukocyte subpopulations, soluble galectin-9 or anatomical pathology.
In summary, single intravenous infusion administration of 30, 100 and 200mg/kg G9.2-17IgG4 to cynomolgus monkeys was tolerated without adverse findings. Thus, under the conditions of this study, the unoeheared adverse reaction level (NOAEL) was 200mg/kg, which is the highest dose level evaluated. The study design is shown in table 18.
TABLE 18 design of experiment
a Group 4 was administered 1 circumferentially after group 1 to group 3 administration.
a Group 4 was administered 1 circumferentially after group 1 to group 3 administration.
During the study, the vehicle and test article were administered by IV infusion through a catheter placed percutaneously in the saphenous vein for 30 minutes. Dosage levels were 30, 100 and 200mg/kg and were administered at a dosage volume of 20 mL/kg. The control group received vehicle in the same manner as the treatment group.
Animals were placed in sling-restraining devices during dosing. The vehicle or test article is administered based on recent body weight and using an infusion pump and sterile disposable syringe. The dosing syringe was filled with the appropriate volume of vehicle or test (20 mL/kg, 2mL additional). At the completion of dosing, animals were removed from the infusion system. The weight of each dosing syringe was recorded before the beginning and end of each infusion to determine dose liability.
Detailed clinical observations
Animals were removed from the cages and each animal was subjected to detailed clinical examinations on day 1 at 1 hour and 4.5 hours after the start of infusion (SOI), after which they were performed once daily during the study period. Animals were removed from the cages and each animal was subjected to detailed clinical examinations on day 1 at 1 hour and 4.5 hours after the start of infusion (SOI), after which they were performed once daily during the study period. Body weights of all animals were measured and recorded weekly at the time of transfer, prior to randomization, day-1 and during the study.
Clinical pathology assessment (hematology, coagulation and clinical chemistry) was performed at all animal pre-trials and at day 1 (pre-dosing), day 3, day 8 and day 21. Additional samples for determining hematology parameters and peripheral blood lymphocyte and cytokine analysis samples were collected 30 minutes after SOI (immediately after infusion) and 4.5, 8.5, 24.5, and 72.5 hours (relative to day 1). Bone marrow smears were collected and stored.
Blood samples (approximately 0.5 mL) were collected from all animals via femoral vein for determination of serum concentration of the test sample (see table 19) (bias see appendix 1). Animals were not fasted prior to blood collection except for the interval following the fasted for clinical pathology collection.
TABLE 19 biological analysis sample collection time Table
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X = collect sample.
a : only animals of group 1 were analyzed for test contents at the time point 0.583 hours after SOI. Additional time points may be analyzed at the discretion of the study taker.
For treatment, blood samples were collected in additive-free unobstructed microtubes and centrifuged at controlled room temperature within 1 hour after collection. The resulting serum was divided into 2 approximately equal aliquots and placed in pre-labeled freezer tubes. All aliquots were stored frozen at-60 ℃ to-90 ℃ within 2 hours after collection.
All euthanized animals were evaluated for post-mortem study at the scheduled necropsy.
Necropsy was performed under procedures approved by a veterinary pathologist. External abnormalities of the animals, including accessible bumps, were carefully examined. The skin was reflected from the ventral midline incision and any subcutaneous mass was identified and correlated with pre-mortem findings. Check if there is abnormality in abdominal cavity, thoracic cavity and cranial cavity. The organ is removed, inspected, and placed in fixative as needed. All designated tissues except the eye (including optic nerve) and testes were fixed in Neutral Buffered Formalin (NBF). Eyes (including optic nerve) and testes were placed in modified davison fixative, then transferred to 70% ethanol for up to three days, before final placement in NBF. Formalin is injected into the lungs through the trachea. Whole tissues and organs were collected from all animals.
The body weight of all animals and the organ weight specified by the protocol were recorded at the scheduled necropsy, and the appropriate organ weight ratio (relative to body weight and brain weight) was calculated. The paired organs were weighed together. The total weight of thyroid and parathyroid glands was collected.
Results
All animals survived to the scheduled necropsy on day 22. No clinical or veterinary results related to the test sample were noted in the treated animals. During the treatment or recovery period, no test-related effects on body weight were observed in the treated animals. At any dose level at any time interval, there was no G9.2-17IgG4 related effect on hematological endpoints in either gender.
At any dose level at any time interval, there was no G9.2-17IgG4 related effect on clotting time (i.e., activated partial thromboplastin time [ APTT ] and prothrombin time) or fibrinogen concentration in either gender. All fluctuations between individual coagulation values are considered sporadic, consistent with biological and procedural related changes, and/or negligible in magnitude, and unrelated to G9.2-17IgG4 administration.
At any dose level at any time interval, there was no G9.2-17IgG4 related effect on clinical chemistry endpoints in either gender. All fluctuations between individual clinical chemistry values are considered sporadic, consistent with biological and procedural related changes, and/or negligible in magnitude, and unrelated to G9.2-17IgG4 administration.
At any dose level at any time interval, there was no G9.2-17IgG 4-related effect on cytokine endpoint in either gender. All fluctuations between individual cytokine values were considered sporadic, consistent with biologically and procedural related changes, and/or negligible in magnitude, and unrelated to G9.2-17IgG4 administration.
Examination of the general necropsy results showed findings that were not considered relevant to the test article. Organ weight changes not considered to be related to the test article. There were no test article related changes.
In summary, single intravenous infusion administration of 30, 100 and 200mg/kg G9.2-17IgG4 to cynomolgus monkeys was tolerated without adverse findings. Thus, under the conditions of this study, the unoeheared adverse reaction level (NOAEL) was 200mg/kg, which is the highest dose level evaluated.
Animals were removed from the cages and each animal was subjected to detailed clinical examinations on day 1 at 1 hour and 4.5 hours after the start of infusion (SOI), after which they were performed once daily during the study period.
EXAMPLE 5 intravenous infusion study of G9.2-17 in cynomolgus monkey
The objective of this study was to further characterize the toxicity and toxicology of different doses of test G9.2-17 (igg 4 monoclonal antibody conjugated to galectin 9) in cynomolgus monkeys after 30 min Intravenous (IV) infusion once a week for 5 weeks, and evaluate the reversibility, progression or delayed appearance of any changes to after 3 weeks of recovery.
Design of experiment
Table 20 summarizes the study design.
TABLE 20 design of experiment
a Based on recent actual weight measurements.
Animals used in the study (cynomolgus monkeys) were assigned to study groups according to standard, weight, randomization procedure (aimed at achieving similar group average weights). Male and female were randomized separately. The body weight assigned to the animals studied was within + -20% of the average body weight per sex.
Formulations lacking G9.2-17 ("vehicle") or comprising G9.2-17 ("test") were administered to animals by 30 minute IV infusion once a week for 5 weeks (day 1, day 8, day 15, day 22, and day 29) during the study. Dosage levels were 0, 100 and 300 mg/kg/dose and were administered at a dosage volume of 10 mL/kg. The control animal group received vehicle in the same manner as the treatment group. Doses were administered via saphenous vein through a percutaneously placed catheter, and a new sterile disposable syringe was used for each administration. Dose accountability was measured and recorded before and at the end of dosing on the pharmacokinetic sample collection days (day 1, day 15 and day 29) to ensure that ±10% of the target dose was administered. Individual doses are based on recent body weight. The last dosing site was marked for collection at end-stage and convalescent necropsy. All doses were administered within 8 hours after the test preparation.
The animals were subjected to a survival program, and measurements, as exemplified below.
Electrocardiographic examination was performed on all animals. Care was taken to avoid causing excessive excitation of the animals prior to recording the Electrocardiogram (ECG) to minimize extreme fluctuations or artifacts in these measurements. Standard ECG (10 leads) was recorded at 50 mm/s. Using the appropriate leads, RR, PR and QT intervals and QRS duration are measured and heart rate is determined. The corrected QT (QTc) interval is calculated using a program based on the method described by Bazett (1920). All traces were evaluated and reported by the consulting veterinary cardiologist.
To aid in continuity and reliability, functional observational test combination (FOB) evaluations were performed by two independent raters for all occasions, consisting of detailed home cage and open area neurobehavioral evaluations (Gauvin and Baird, 2008). Each technician scored monkeys independently (without sharing results with each other) for each home and out-of-home score, and then assessed whether individual scores were consistent with the scores of their partners after the trial was completed. Each animal was subjected to a FOB evaluation prior to dosing (either on day-9 or day 8) to establish baseline differences, and 2 to 4 hours from the start of infusion on day 1 and day 15, and prior to end-stage and convalescent necropsy. Including but not limited to, assessment of activity level, posture, tear flow, salivation, tremor, twitching, spontaneous contractions, notch behavior, facial muscle movements, eyelid closure, pupillary response, response to stimuli (visual, auditory and food), body temperature, chaddock and Babinski reflex, proprioception, paresis, ataxia, range finding inaccuracy and slope assessment, movement and gait.
Blood pressure, including systolic, diastolic and mean arterial pressure, was measured and recorded for each animal. Blood pressure measurements were reported using three readings with Mean Arterial Pressure (MAP) within 20 mmHg.
The respiratory rate of each animal was measured and recorded 3 times at animal/collection intervals by visual assessment according to test facility SOP. The average of 3 collections is reported.
All animals were subjected to clinical pathology assessment (e.g., immunophenotyping and cytokine assessment) at predetermined time intervals. Bone marrow smears were collected and stored. Blood samples (approximately 0.5 mL) were collected from all animals via the femoral vein for determination of serum concentration of the test sample. Animals were not fasted prior to blood collection except for the interval following the fasted for clinical pathology collection. At the end of the study (day 36 or day 50), animals were euthanized and tissues were collected for histological treatment and microscopic evaluation.
Soluble galectin-9 was evaluated as follows. Blood samples (approximately 1 mL) were collected from all animals intravenously via the femoral vein 24 hours before dosing on day 1, day 8, day 15 and day 29 and beginning of infusion and before end-stage and/or convalescent necropsy for determination of serum soluble galectin-9. Animals were not fasted prior to blood collection except for the interval following the fasted for clinical pathology collection.
Soluble galectin-9 samples were treated as follows. Blood samples were collected in additive-free unobstructed tubes, allowed to coagulate at ambient temperature, and centrifuged at ambient temperature. The resulting serum was split into 2 aliquots (100 μl in aliquot 1 and the remainder in aliquot 2) and placed in pre-labeled freezer tubes. All aliquots were flash frozen on dry ice within 2 hours after collection and stored frozen at-60 to 90 ℃.
All the results shown in the reported tables are calculated from the original data rounding procedure using non-rounded values and may not be accurately reproduced from the single data provided.
Results
● Mortality rate of
All animals survived to a predetermined end-stage necropsy on day 36 and a convalescent necropsy on day 50.
● Detailed clinical and veterinary observations
During the treatment or recovery period, no clinical or veterinary observations related to the test article were noted in the treated animals.
● Functional observation test combination
During the treatment or recovery period, no test-related FOB observations were noted in the treated animals.
● Body weight and weight gain
During the treatment or recovery period, no test-related effects on body weight and body weight gain were noted in the treated animals.
● Ophthalmic examination
During the treatment or recovery period, no test-related effects on the ocular examination were noted in the treated animals.
● Blood pressure value
During the treatment or recovery period, no test-related effects on blood pressure values were noted in the treated animals.
● Respiratory rate values
During the treatment or recovery period, no test-related effects on respiratory rate values were noted in the treated animals.
● Electrocardiogram system
During the treatment or recovery period, no test-related effects on the electrocardiographic assessment were noted in the treated animals.
● Hematology
At any dose level at any time point, there was no G9.2-17 related effect on hematological parameters in either gender.
● Coagulation
At any dose level at any time point, there was no G9.2-17 related effect on coagulation parameters in either gender.
● Clinical chemistry
At any dose level at any time point, there was no G9.2-17 related effect on clinical chemistry parameters in either gender.
● Urine analysis
During the middle of 13 weeks, no G9.2-17 related changes were observed among the urinalysis parameters of either sex at any dose level.
● Cytokines and methods of use
No G9.2-17 related clear effect on cytokines was observed at any dose level or time point.
● Peripheral Blood Leukocyte Analysis (PBLA)
At any dose level at any time point, there was no G9.2-17 related effect on PBLA endpoint in either gender.
● Bioassay, galectin-9 and toxicokinetics evaluation
After dose administration, G9.2-17 was quantifiable in all cynomolgus monkey samples from all animals administered G9.2-17. No measurable amount of G9.2-17 was detected in the control cynomolgus monkey sample. In all cynomolgus monkey samples from all animals, soluble galectin-9 was quantifiable. In all serum samples obtained on day 1 from most of the G9.2-17 treated animals and on days 1 and 29 from control animals prior to dosing, the G9.2-17 serum concentrations were below bioassay quantification limit (LLOQ <0.04 ug/mL).
● Gross pathology and organ weight
There were no clear macroscopic observations related to the test article in the main study or convalescent animals. For animals in the main study or convalescence period, there was also no sample related organ weight change.
● Histopathology
There was no clear microscopic observation of the sample correlation.
In summary, once weekly intravenous infusion administration of 100 and 300mg/kg G9.2-17 to cynomolgus monkeys was tolerated for 5 weeks with no adverse findings.
EXAMPLE 6 intravenous infusion study of G9.2-17 in Sprague Dawley rats
The aim of this study was to evaluate the potential toxicity of different doses of G9.2-17 (an IgG4 human monoclonal antibody against galectin-9) when administered by intravenous infusion to Sprague Dawley rats (once a week for 4 weeks followed by a recovery period after 3 weeks of administration). In addition, the pharmacokinetic profile of G9.2-17 was determined.
Design of experiment
Table 21 summarizes the study design.
Table 21: study design
a Individual dose volumes were calculated from the most recent body weight.
b SSD animals: 3 animals/sex/group were used for TK collection, only after a single dose administration on day 1.
One hundred eighty six animals (Sprague Dawley rats) were randomly assigned to the treatment group by body weight. The control/vehicle, formulation buffer for test article and test article G9.2-17 were administered by a single IV injection in the tail vein at dosage levels of 0, 100 and 300mg/kg on days 1, 8, 15, 22 and 29. The test article was administered once to animals assigned to the SSD subgroup at dose levels of 100 and 300mg/kg on day 1.
Starting from the next day of environmental adaptation, a clinical observation was made every morning before cleaning the room. Mortality checks were performed twice daily to assess general animal health. Food consumption was estimated by weighing the food supply and the remaining amount in the container once a week. Average grams (g)/animal/day was calculated from weekly food consumption. Body weight was measured prior to randomization, on day-1, then weekly throughout the study, and on the day of each necropsy. Functional observation test combination (FOB) observations were recorded for SSB animals at approximately 24 hours after dose administration on days 1, 35 and 49. Urine was collected overnight using a metabolic cage. Samples were obtained on day 36 and day 50.
Animals were fasted overnight prior to each series collection including samples for serum chemistry. In these cases, the relevant clinical pathology assessment was from fasted animals. Blood is collected from the jugular vein of a restrained awake animal or from the vena cava of an anesthetized animal (at the end).
Parameters assessed during the survival check of the study included clinical observations, food consumption, body weight, functional observations and combinations of tests. Blood samples were collected at selected time points for clinical pathology (hematology, coagulation and serum chemistry) analysis. Urine samples were collected for urine analysis. Blood samples were also collected at selected time points for toxico-kinetic (TK), immunogenicity (e.g., anti-drug antibodies or ADA), and cytokine analysis. The animals were necropsied on day 36 and day 50. At each necropsy, gross observations and organ weights were recorded and tissues were collected for microscopy.
Results
Survival check
Mortality rate of: no abnormal clinical observations or body weight changes were noted in the animals during the study.
Clinical observation:no G9.2-17 related clinical observations were noted during the study.
Food consumption/body weight: no changes in G9.2-17 related food consumption, body weight or weight gain were noted during the study.
Clinical pathology: no G9.2-17 related changes were noted in the clinical pathology parameters.
Cytokine analysis: there were no G9.2-17 related changes in serum concentrations of IL-2, IL-4, IFN-gamma, IL-5, IL-6, IL-10 and/or TNF-alpha, MCP-1 and MIP-1 b.
General pathology: there were no general observations related to G9.2-17. Furthermore, there was no change in absolute or relative organ weights associated with G9.2-17.
Histopathology: there was no histological findings associated with G9.2-17.
In summary, intravenous G9.2-17 administration of a total of 5 doses once a week to Sprague Dawley rats is generally well tolerated. There were no G9.2-17 related clinical observations, food consumption, body weight, FOB parameters, clinical pathology, cytokines, gross observations, or changes in organ weight.
EXAMPLE 7 inhibition of polarization and repolarization of M2 macrophages
Macrophages play an indispensable role in the immune system, and have a decisive function in both innate and acquired immunity. M1 macrophages are generally thought to be potent effector cells that can kill tumor cells, while M2 polarized macrophages express a range of cytokines, chemokines and proteases to promote angiogenesis, lymphangiogenesis, tumor growth, metastasis and immunosuppression (Sica et al, 2008;Semin.Cancer Biol.2008;18:349-355). In M2 macrophages, production of anti-inflammatory cytokines such as TGF-beta and IL-10 is enhanced (Martinez et al, front biosci.2008, 1 month; 13:453-61; mantovani et al, trends Immunol 2002, 11 month; 23 (11): 549-55; zhang et al, J Hematol Oncol 10,58 (2017)). Given that macrophages constitute a key component of the host immune response, inhibition of polarization or repolarization of M2 macrophages is an important therapeutic consideration in tumor immunotherapy (Poh and Ernst, front oncol.2018, day 3, month 12; 8:49).
Cd14+ monocytes were isolated using whole blood from three healthy human donors. Monocytes were allowed to differentiate into macrophages in X-VIVO-15 medium (Lonza) in 10cm tissue culture dishes for 7 days. Differentiated macrophages are used directly to assess inhibition of polarization, or they are cryopreserved and used for repolarization assays at a later time. Prior to use in the assay, M0 macrophages were phenotyped.
Macrophage polarization was evaluated using two different polarization mixtures: a mixture containing IL-4 and IL-13, while the second contains only gal-9. The effect on M2 polarization was tested by adding G9.2-17 directly to one of these mixtures and incubating with macrophages for 48 hours. The effect of G9.2-17 on repolarization of M2 macrophages was tested via addition to M2 polarized macrophages.
The polarization state was identified by measuring secretion of IL-10 (repolarization) or TGF- β1 (polarization and inhibition of repolarization). These factors were quantified in cell culture supernatants using CytoMetric Bead Arrays according to the manufacturer's protocol.
Representative data from one donor showing the effect of G9.2-17 on the polarization of fresh monocyte-derived macrophages is in fig. 5. All donor macrophages showed similar results with reduced TGF- β1 secretion after incubation with G9.2-17 compared to isotype matched control or untreated cells. FIG. 5 shows the effect of previously frozen macrophages on TGF- β1 secretion after incubation with G9.2-17 or isotype matched controls. Treatment with 20ng/mL of the polarizing mixture significantly induced TGF- β1 secretion, whereas G9.2-17 treatment eliminated the IL-4/IL-13 dependent increase in TGF- β1 secretion. FIG. 6 shows the effect of IL-10 secretion on repolarization of cryopreserved macrophages. In the presence of both types of polarisation mixtures, treatment with G9.2-17 resulted in reduced levels of secreted IL-10 and TGF-b1 in all donors compared to untreated and IgG4 isotype control antibody controls.
This assay demonstrates that G9.2-17 can potently inhibit TGF- β1 and IL-10 at a concentration of 20 μg/ml.
Example 8 measurement of biomarkers
Multiple immunofluorescence (mIF) techniques are performed on clinical tissue from a patient. The mIF assay consisted of 10 rounds of staining, each round staining and imaging two biomarkers for a total of ten rounds. For each round, one antibody is conjugated with one of the two fluorescent dyes that will allow imaging of the biomarker, such that each round images the two biomarkers. Biomarkers are stained, imaged, and then the signal quenched to allow further staining and imaging rounds to occur without exuding competing signals. When staining and imaging of the complete 19-marker panel is complete, the positives of each biomarker on the cells are classified by a deep learning algorithm trained to detect positive signals. When the analysis is complete, various data are generated, including the density and raw count of positive cells for each biomarker and co-expression of interest. Biomarkers include CD3, CD4, CD8, CD45RO, foxP3, CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, arginase 1, granzyme B, ki67, PD1, PD-L1, F4/80, ly6G/C, and PanCK.
EXAMPLE 9 evaluation of mouse galectin-9 in plasma by ELISA
This study evaluated galectin 9 in plasma of female C57BL/6 mice (the same mouse model as example 1 above) in situ pancreatic cancer xenograft model mPA 6115. Mice were assigned to multiple groups and treated according to the study design shown in table 3 above. According to the protocol, plasma samples were collected from the retroorbital sinus on day 3 prior to the 1 st dose, and for euthanized mice dying (post dose), by cardiac puncture at the time of sacrifice, for all mice of groups 1-6 (pre dose) and 10 non-tumor bearing mice in group 7 (tumor implant on day 0) that were transplanted with tumors.
Plasma samples were analyzed for galectin-9 levels by ELISA according to the following procedure:
1. all reagents and samples were left at room temperature (18-25 ℃) prior to use. All standards and samples were recommended to be run at least in duplicate.
2. According to the experiment, removable 8-hole slats were marked, where appropriate.
3. Mu.l of each standard and prepared sample were added to the appropriate wells. The wells were covered and incubated for 2.5 hours at room temperature with gentle shaking.
4. The solution was discarded and washed 4 times with 1X wash solution. Washing was performed by filling each well with wash buffer (300 μl) using a multichannel pipette or an automatic washing machine. Complete removal of liquid in each step is critical for good performance. After the last wash, any remaining wash buffer is removed by aspiration or decantation. The plate was inverted and blotted dry with a clean paper towel.
5. 100 μl of 1X prepared biotinylated antibody (reagent preparation step 3) was added to each well. Incubate with gentle shaking at room temperature for 1 hour.
6. The solution was discarded. The wash as in step 4 is repeated.
7. Mu.l of the prepared streptavidin solution was added to each well. Incubate for 45 min at room temperature with gentle shaking.
8. The solution was discarded. The wash as in step 4 is repeated.
9. Mu.l TMB one-step substrate reagent was added to each well. Incubate for 30 min at room temperature with gentle shaking in the absence of light.
10. Mu.l of stop solution was added to each well. Read immediately at 450 nm.
The results demonstrate that once tumors were transplanted in situ, galectin-9 serum levels in the mPA6115 mouse model increased, consistent with observations in pancreatic adenocarcinoma patients. This study demonstrated that galectin-9 serum levels were significantly increased in animals grown in situ with pancreatic ductal adenocarcinoma. This means that the source of this galectin-9 is indeed tumor tissue, further supporting the therapeutic approach of blocking galectin-9 in the context of this disease.
Equivalent scheme
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the following claims.
Although several inventive embodiments have been described and illustrated herein, various other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein will be apparent to those of ordinary skill in the art, and each such variation and/or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations depend upon the specific application for which the teachings of the invention is used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, any combination of two or more such features, systems, articles, materials, kits, and/or methods is included within the scope of the present disclosure.
All definitions, as defined and used herein, should be understood to have precedence over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference to each of the cited subject matter, and in some cases, may encompass the entire document.
The indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.
As used herein in the specification and claims, the phrase "and/or" should be understood to mean "either or both" of the elements connected, i.e., the elements are in some cases present in combination and in other cases present separately. The use of "and/or" listed elements should be interpreted in the same manner, i.e. "one or more" of the elements connected. In addition to the elements specifically identified by the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, reference to "a and/or B" when used in conjunction with an open language such as "comprising" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment may refer to B alone (optionally including elements other than a); in another embodiment, a and B (optionally including other elements) may be referred to; etc.
As used herein in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items are separated in a list, "or" and/or "should be understood to be inclusive, i.e., including at least one, but also including more than one of the elements or list of elements, and optionally, including additional unlisted items. Only the opposite terms, such as "only one of … …" or "exactly one of … …" or "consisting of … …" when used in a claim, will be referred to as comprising exactly one element of a plurality or list of elements. In general, the term "or" as used herein should be interpreted to mean exclusive choice (i.e., "one or the other but not both") only when crowned with exclusive terms such as "either," one of … …, "" only one of … …, "or" exactly one of … …. "consisting essentially of … …," when used in the claims, shall have its ordinary meaning as used in the patent statutes.
As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combination of elements in the list of elements. The definition also allows that elements other than the specifically identified elements within the list of elements to which the phrase "at least one" refers may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of a and B" (or, equivalently, "at least one of a or B," or, equivalently, "at least one of a and/or B") can refer in one embodiment to at least one (optionally including more than one) a without B present (and optionally including elements other than B); in another embodiment at least one (optionally including more than one) B is absent a (and optionally including elements other than a); in another embodiment at least one (optionally including more than one) a and at least one (optionally including more than one) B (and optionally including other elements); etc.
It should also be understood that the order of steps or acts of any method claimed herein that includes more than one step or act is not necessarily limited to the order in which the steps or acts of the method are recited, unless explicitly stated to the contrary.
Sequence listing
<110> PureTech LYT company (PureTech LYT, inc.)
<120> combination of anti-galectin-9 antibody and chemotherapeutic agent for use in cancer therapy
<130> 112174-0212 (NP010WO1)
<140> not yet allocated
<141> along with the submission
<150> US 63/313,883
<151> 2022-02-25
<150> US 63/193,381
<151> 2021-05-26
<150> US 63/182,519
<151> 2021-04-30
<160> 35
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 1
Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala
1 5 10
<210> 2
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 2
Ser Ala Ser Ser Leu Tyr Ser
1 5
<210> 3
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 3
Gln Gln Ser Ser Thr Asp Pro Ile Thr
1 5
<210> 4
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 4
Phe Thr Val Ser Ser Ser Ser Ile His
1 5
<210> 5
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 5
Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 6
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 6
Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp Tyr
1 5 10 15
<210> 7
<211> 124
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 7
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 108
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Asp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 9
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 9
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser
20
<210> 10
<211> 330
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 10
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 11
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 11
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 12
<211> 330
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 12
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 13
<211> 327
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 13
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Pro Gly Lys
325
<210> 14
<211> 327
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 14
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Pro Gly Lys
325
<210> 15
<211> 214
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ser Thr Asp Pro Ile
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 16
<211> 454
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 16
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 17
<211> 454
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys
450
<210> 18
<211> 451
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 19
<211> 451
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 20
<211> 327
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 20
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 21
<211> 327
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 21
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly Lys
325
<210> 22
<211> 451
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 22
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Leu Gly Lys
450
<210> 23
<211> 451
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Leu Gly Lys
450
<210> 24
<211> 329
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 24
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 25
<211> 329
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 25
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly
325
<210> 26
<211> 326
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 26
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Pro Gly
325
<210> 27
<211> 326
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 27
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly
325
<210> 28
<211> 326
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 28
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Pro Gly
325
<210> 29
<211> 326
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 29
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr
65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
195 200 205
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
225 230 235 240
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
305 310 315 320
Leu Ser Leu Ser Leu Gly
325
<210> 30
<211> 453
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 30
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly
450
<210> 31
<211> 453
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 31
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly
450
<210> 32
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 32
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 33
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 33
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Leu Gly
450
<210> 34
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 34
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly
450
<210> 35
<211> 450
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> Synthesis
<400> 35
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Ser
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Trp Ser Tyr Pro Ser Trp Trp Pro Tyr Arg Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
210 215 220
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
260 265 270
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Leu Gly
450

Claims (46)

1. A method for treating a solid tumor comprising administering to a subject in need thereof an effective amount of an antibody that binds human galectin-9 (anti-Gal 9 antibody) and an effective amount of one or more chemotherapeutic agents; wherein the anti-Gal 9 antibody has the same heavy chain Complementarity Determining Regions (CDRs) and the same light chain CDRs as antibodies G9.2-17, and wherein the subject has one or more of the following features:
(i) No resectable cancer;
(ii) Is not infected by SARS-CoV-2; and
(iii) There was no active brain or leptomeningeal metastasis.
2. The method of claim 1, wherein said anti-Gal 9 antibody is administered to said subject at a dose of about 0.2mg/kg to about 32mg/kg once every two weeks to once every 6 weeks, optionally once every two weeks.
3. The method of claim 2, wherein said anti-Gal 9 antibody is administered to said subject at a dose of about 0.2mg/kg to about 16mg/kg once every two weeks to once every six weeks, optionally once every two weeks.
4. The method of claim 3, wherein the anti-Gal 9 antibody is administered to said subject at a dose of about 0.2mg/kg, about 0.6mg/kg, about 0.63mg/kg, about 2mg/kg, about 4mg/kg, about 6mg/kg, about 6.3mg/kg, about 8mg/kg, about 10mg/kg, about 12mg/kg, or about 16mg/kg once every two weeks to once every six weeks, optionally once every two weeks.
5. The method of claim 1, wherein said anti-Gal 9 antibody is administered to said subject at a dose of about 650mg to about 1120mg once every two weeks to once every six weeks, optionally once every two weeks.
6. The method of claim 5, wherein said anti-Gal 9 antibody is administered to said subject at a dose of about 650mg to about 700mg once every two weeks to six weeks, optionally once every two weeks, or once every two weeks to six weeks, optionally once every two weeks at a dose of about 1040mg to about 1120 mg.
7. A method for treating a solid tumor comprising administering to a subject in need thereof an effective amount of an antibody that binds human galectin-9 (anti-Gal 9 antibody) and an effective amount of one or more chemotherapeutic agents;
wherein the anti-Gal-9 antibody comprises:
(a) A light chain comprising a light chain variable region (VL) comprising a Light Chain (LC) complementarity determining region 1 (CDR 1) comprising the amino acid sequence of SEQ ID NO. 1, a LC complementarity determining region 2 (CDR 2) comprising the amino acid sequence of SEQ ID NO. 2 and a LC complementarity determining region 3 (CDR 3) comprising the amino acid sequence of SEQ ID NO. 3, and
(b) A heavy chain comprising a heavy chain variable region (VH) comprising Heavy Chain (HC) complementarity determining region 1 (CDR 1) comprising the amino acid sequence of SEQ ID No. 4, HC complementarity determining region 2 (CDR 2) comprising the amino acid sequence of SEQ ID No. 5 and HC complementarity determining region 3 (CDR 3) comprising the amino acid sequence of SEQ ID No. 6; and is also provided with
Wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 0.2-32mg/kg once a week, optionally wherein the anti-galectin-9 antibody is administered to the subject at a dose of about 10mg/kg to about 16mg/kg once a week.
8. The method of claim 7, wherein the anti-Gal-9 antibody is administered to the subject at a dose of 10mg/kg or 16mg/kg once a week.
9. The method of claim 8, wherein the anti-Gal-9 antibody is administered to said subject at a dose of about 650mg to about 1120mg once a week.
10. The method of claim 9, wherein said anti-Gal 9 antibody is administered to said subject at a dose of about 650mg to about 700mg once a week or at a dose of about 1040 to about 1120mg once a week.
11. The method of any one of claims 1-10, wherein the solid tumor is an metastatic solid tumor.
12. The method of claim 11, wherein the solid tumor is Pancreatic Ductal Adenocarcinoma (PDAC), colorectal cancer (CRC), hepatocellular carcinoma (HCC), cholangiocarcinoma (CAA), renal Cell Carcinoma (RCC), urothelial carcinoma, head and neck cancer, breast cancer, lung cancer, or Gastrointestinal (GI) solid tumor.
13. The method of claim 12, wherein the subject is free of locally advanced PDACs that are free of distant organ metastatic deposition.
14. The method of any one of claims 1-13, wherein the anti-Gal-9 antibody is administered to the subject by intravenous infusion.
15. The method of any one of claims 1-4, wherein V of said anti-Gal-9 antibody L Comprising the amino acid sequence of SEQ ID NO. 8.
16. The method of any one of claims 1-15, wherein V of said anti-Gal-9 antibody H Comprising the amino acid sequence of SEQ ID NO. 7.
17. The method of any one of claims 1-16, wherein said anti-Gal-9 antibody is a full length antibody.
18. The method of claim 17, wherein the anti-Gal-9 antibody is an IgG1 or IgG4 molecule.
19. The method of claim 18, wherein the anti-Gal-9 antibody is a human IgG4 molecule having an Fc region modified relative to a wild-type human IgG4 counterpart.
20. The method of claim 19, wherein the modified Fc region comprises the amino acid sequence of SEQ ID No. 14.
21. The method of any one of claims 1-20, wherein said anti-Gal-9 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 19 and a light chain comprising the amino acid sequence of SEQ ID No. 15.
22. The method of any one of claims 1-21, wherein the one or more chemotherapeutic agents comprise an antimetabolite, a microtubule inhibitor, or a combination thereof.
23. The method of claim 22, wherein the antimetabolite is gemcitabine and the microtubule inhibitor is paclitaxel, or a combination thereof.
24. The method of claim 23, wherein the paclitaxel is protein-bound paclitaxel.
25. The method of claim 24, wherein the paclitaxel is nanoparticle albumin-bound paclitaxel.
26. The method of any one of claims 1-6 and 11-25, wherein the method comprises a cycle of 28 days, wherein the anti-Gal-9 antibody is administered to the subject on days 1 and 15, and the gemcitabine and paclitaxel are administered to the subject on days 1, 8, and 15.
27. The method of any one of claims 7-25, wherein the method comprises a 28 day cycle, wherein the anti-Gal 9 antibody is administered to the subject on days 1, 8, 15, and 22, and the gemcitabine and paclitaxel are administered to the subject on days 1, 8, and 15.
28. The method of any one of claims 23-27, wherein the amount is 125mg/m 2 Administering the paclitaxel intravenously to the subject.
29. The method of claim 28, wherein the concentration is 1000mg/m 2 Administering the gemcitabine to the subject.
30. The method of any one of claims 1-29, wherein the subject is a human patient.
31. The method of any one of claims 1-30, wherein the subject comprises a galectin-9 positive cancer cell or immune cell.
32. The method of any one of claims 1-31, wherein the subject has elevated galectin-9 levels relative to a control value.
33. The method of claim 32, wherein the subject has elevated serum or plasma levels of galectin-9 relative to the control value.
34. The method of any one of claims 1-33, wherein the subject has received at least one line of systemic anticancer therapy.
35. The method of any one of claims 1-34, wherein the subject has not received prior therapy involving gemcitabine and/or paclitaxel or has received prior therapy involving gemcitabine and/or paclitaxel at least six months prior to administration of the anti-Gal 9 antibody.
36. The method of any one of claims 1-35, wherein the subject is examined for one or more of the following characteristics prior to, during, and/or after the treatment:
(a) One or more tumor markers in a tumor biopsy sample from the subject, optionally wherein the one or more tumor markers comprise CA15-3, CA-125, CEA, CA19-9, and/or alpha fetoprotein;
(b) Cytokine profile; and
(c) Galectin 9 level.
37. The method of any one of claims 1-36, wherein the method further comprises monitoring the subject for the occurrence of one or more adverse effects.
38. The method of claim 37, wherein the one or more adverse effects comprise liver injury, blood toxicity, neurotoxicity, skin toxicity, gastrointestinal toxicity, or a combination thereof.
39. The method of claim 37 or 38, further comprising reducing the dose of said anti-Gal 9 antibody, the dose of said one or more chemotherapeutic agents, or both, when adverse effects are observed.
40. The method of claim 39, wherein administration of the paclitaxel is stopped when the subject's aspartate Aminotransferase (AST) level is greater than 10 x upper normal limit (ULN), bilirubin level is greater than 5 x ULN, or both.
41. The method of claim 40, wherein the method comprises reducing the dose of the anti-galectin-9 antibody, the dose of gemcitabine, the dose of paclitaxel, or a combination thereof when moderate to severe liver injury is observed.
42. The method of claim 41, wherein said method comprises reducing the dosage of or terminating administration of said anti-Gal 9 antibody, said gemcitabine, said paclitaxel, or a combination thereof when severe hematologic toxicity, neurotoxicity, dermatotoxicity, and/or gastrointestinal toxicity is observed.
43. The method of claim 41 or 42, wherein the dose of paclitaxel is reduced to 100mg/m 2 -75 mg/m 2 。
44. The method of any one of claims 41-43, wherein the dose of gemcitabine is reduced to 800mg/m 2 -600 mg/m 2 。
45. The method of any one of claims 1-44, wherein a plurality of doses of said anti-Gal 9 antibody are administered to said subject, and a later dose is higher than an earlier dose.
46. The method of any one of claims 1-44, wherein a plurality of doses of said anti-Gal-9 antibody are administered to said subject, and a later dose is lower than an earlier dose.
CN202280045695.8A 2021-04-30 2022-04-29 Combination of anti-galectin-9 antibodies and chemotherapeutic agents for use in cancer therapy Pending CN117580589A (en)

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US63/182,519 2021-04-30
US63/193,381 2021-05-26
US202263313882P 2022-02-25 2022-02-25
US63/313,882 2022-02-25
PCT/US2022/027142 WO2022232653A1 (en) 2021-04-30 2022-04-29 Combination of anti-galectin-9 antibodies and chemotherapeutics for use in cancer therapy

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