Combinations of anti-CTLA-4 antibodies and anti-PD-1 antibodies and their therapeutic use

Abstract

This specification provides a combination of anti-CTLA-4 antibody and anti-PD-1 antibody, as well as its use for cancer treatment.

Description

Technical Field

[0001] The present invention relates to combinations of anti-CTLA-4 antibodies and anti-PD-1 antibodies and their use for cancer treatment. Reference to Sequence Listing

[0002] This application includes a sequence listing submitted in XML format by the Patent Center, the full text of which is incorporated herein by reference. The name of the file created on April 25, 2024 is Sequence-Listing-111005-0606-01PC00.xml and it has a size of 37 kb.

Background Art

[0003] Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) interacts with B7-1 (CD80) and B7-2 (CD86) to ensure proper function of regulatory T cells and protect the host against autoinflammatory diseases. Anti-CTLA-4 monoclonal antibodies (mAbs) such as ipilimumab have demonstrated potent and broad cancer immunotherapy effects (CITE) in various preclinical models and early clinical trials and are currently approved by the US FDA for clinical use both as monotherapy and as part of combination therapy with nivolumab (anti-PD-1). However, compared to anti-PD-1 / PD-L1 antibodies, CTLA-4 targeting in cancer patients has been less successful. Direct comparisons have revealed that the response rate of melanoma patients treated with ipilimumab is consistently lower than that of anti-PD-1 antibodies. Thus, anti-PD-1 / PD-L1 antibodies have obtained approval for clinical use in the rapid expansion of indications, while monotherapy with anti-CTLA-4 antibodies has failed in multiple Phase III clinical trials except for melanoma.

[0004] Furthermore, CTLA-4 monotherapy has a higher incidence of immune-related adverse events (irAEs) than anti-PD-1 / PD-L1 therapy. In melanoma patients receiving neoadjuvant therapy with ipilimumab and nivolumab combination, the rate of severe irAEs (grades 3 and 4) reached 73–90%. This potent irAE further limits the doses accepted by cancer patients. Nevertheless, combinations with anti-PD-1 have resulted in significantly improved response rates and patient survival rates in multiple cancer types. Moreover, anti-CTLA-4 antibodies can induce long-lasting immunity in cancer patients. Therefore, while CTLA-4 holds an important immunotherapy target, major challenges remain in improving the safety, efficacy, and therapeutic effects of anti-CTLA-4 mAbs.

[0005] Generally, the combination of anti-CTLA-4 antibodies and anti-PD-1 / PD-L1 antibodies results in increased antitumor activity. The nivolumab + ipilimumab combination has proven effective and received FDA approval for the treatment of patients with metastatic melanoma, renal cell carcinoma, or high microsatellite instability (MSI-high) colorectal cancer. For example, in the Phase III trial CheckMate-067, the nivolumab + ipilimumab combination was compared to nivolumab or ipilimumab monotherapy in previously untreated metastatic melanoma. At a minimum follow-up of 60 months, the median overall survival was longer than 60.0 months (median not reached) in the nivolumab + ipilimumab group compared to 19.9 months in the ipilimumab group, and was 36.9 months in the nivolumab group (hazard ratio for death in nivolumab + ipilimumab compared to ipilimumab: 0.52; hazard ratio for death in nivolumab compared to ipilimumab: 0.63).

[0006] However, compared to PD-1 nivolumab monotherapy or CTLA-4 ipilimumab monotherapy, the ipilimumab-nivolumab combination is associated with a less favorable safety profile. As expected from the different mechanisms of action of these drugs acting on different lymphocyte subtypes and different receptor sites, their concurrent use, such as 3 mg / kg ipilimumab and 1 mg / kg nivolumab, results in both a higher incidence of immune-related adverse events (irAEs) and a broader scope of application. Grade 3 or 4 treatment-related adverse events occurred in 23% of patients in the nivolumab group, 28% in the ipilimumab group, and 59% in the nivolumab + ipilimumab group, respectively.

[0007] The combination of nivolumab 3 mg / kg every two weeks (q2w) and ipilimumab 1 mg / kg every six weeks (q6w) was tested in patients with advanced / metastatic non-small cell lung cancer (NSCLC) in the CheckMate-227 clinical trial. Among patients with PD-L1 expression levels of ≥1%, the median overall survival was 17.1 months (95% confidence interval [CI], 15.0–20.1) with nivolumab + ipilimumab and 14.9 months (95% CI, 12.7–16.7) with chemotherapy (P=0.007), with 2-year overall survival rates of 40.0% and 32.8%, respectively. The median duration of response was 23.2 months with nivolumab + ipilimumab and 6.2 months with chemotherapy. An overall survival effect was also observed in patients with PD-L1 expression levels of less than 1%, with a median overall survival of 17.2 months (95% CI, 12.8–22.0) for nivolumab + ipilimumab and 12.2 months (95% CI, 9.2–14.3) for chemotherapy. Among all patients in the trial, the median overall survival was 17.1 months (95% CI, 15.2–19.9) for nivolumab + ipilimumab and 13.9 months (95% CI, 12.2–15.1) for chemotherapy. The percentage of patients experiencing grade 3 or 4 treatment-related adverse events in the overall population was 32.8% for nivolumab + ipilimumab and 36.0% for chemotherapy. The high risk of severe toxicity with the combination of the two antibodies limits clinical development. Therefore, there is a need in the art for safer combinations of anti-CTLA-4 and anti-PD-1 antibodies. [Overview of the project]

[0008] This specification provides compositions comprising an anti-CTLA-4 antibody and an anti-PD-1 antibody. The anti-CTLA-4 antibody may comprise (i) a light chain variable region comprising a complementation-determining region (CDR) 1 comprising the sequence described in SEQ ID NO: 1, a CDR2 comprising the sequence described in SEQ ID NO: 3, and a CDR3 comprising the sequence described in SEQ ID NO: 5; and (ii) a heavy chain variable region comprising a CDR1 comprising the sequence described in SEQ ID NO: 6, a CDR2 comprising the sequence described in SEQ ID NO: 9, and a CDR3 comprising the sequence described in SEQ ID NO: 10. The anti-CTLA-4 antibody may comprise a light chain variable region comprising the sequence described in SEQ ID NO: 12 and a heavy chain variable region comprising the sequence described in SEQ ID NO: 16. The anti-CTLA-4 antibody may comprise a light chain comprising the sequence described in SEQ ID NO: 23 and a heavy chain comprising the sequence described in SEQ ID NO: 21.

[0009] An anti-PD-1 antibody may include (i) a light chain variable region including a CDR1 containing the sequence described in SEQ ID NO: 25, a CDR2 containing the sequence described in SEQ ID NO: 26, and a CDR3 containing the sequence described in SEQ ID NO: 27; and (ii) a heavy chain variable region including a CDR1 containing the sequence described in SEQ ID NO: 31, a CDR2 containing the sequence described in SEQ ID NO: 32, and a CDR3 containing the sequence described in SEQ ID NO: 33. An anti-PD-1 antibody may include a light chain variable region including the sequence described in SEQ ID NO: 28 and a heavy chain variable region including the sequence described in SEQ ID NO: 34. An anti-PD-1 antibody may include a heavy chain including IgG4 containing the sequence described in SEQ ID NO: 35 or SEQ ID NO: 36. An anti-PD-1 antibody may include a light chain containing the sequence described in SEQ ID NO: 30 and a heavy chain containing the sequence described in SEQ ID NO: 37. An anti-CTLA-4 antibody may include a light chain containing the sequence described in SEQ ID NO: 23 and a heavy chain containing the sequence described in SEQ ID NO: 21; and the anti-PD-1 antibody may include a light chain containing the sequence described in SEQ ID NO: 30 and a heavy chain containing the sequence described in SEQ ID NO: 37.

[0010] The composition may contain 1 to 50 mg / mL each of anti-CTLA-4 antibody and anti-PD-1 antibody. The anti-CTLA-4 antibody and anti-PD-1 antibody may be combined in the same buffer. The composition may contain 10 mg / mL each of anti-CTLA-4 antibody and anti-PD-1 antibody, or 10 mg / mL or less. The composition may contain a pharmaceutically acceptable carrier. The composition may contain one or more of histidine buffer, α,α-trehalose dihydrate, polysorbate 80 (PS80), and EDTA. The composition may contain 5 to 40 mM histidine buffer, 7.0% to 10.0% (weight / volume) of α,α-trehalose dihydrate, and 0.01 to 0.10 (weight / volume) of PS80. The pH of the composition may be 5.5 to 6.5. The composition may contain 20 mM histidine buffer, 8.8% (weight / volume) α,α-trehalose dihydrate, 0.06% (weight / volume) PS80, and 0.2 mM EDTA·2Na·2H2O, and the pH of the composition may be 6.0.

[0011] This specification provides a method for treating cancer in a subject requiring cancer treatment, which may include administering the composition to the subject. Furthermore, it provides the use of the composition in the manufacture of a pharmacopoeia for the treatment of the cancer, and the composition for use in the treatment of the cancer. The cancer may be a tumor. The cancer may be melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), pancreatic cancer, or triple-negative breast cancer (TNBC). The ovarian cancer may be high-grade serous ovarian cancer, which may be primary peritoneal cancer or fallopian tube cancer. The cancer may be PD-1 resistant.

[0012] The anti-CTLA-4 antibody and the anti-PD-1 antibody, each at a dose of 10 mg / kg, may be administered to the subject, or may be intended to be administered to the subject. The composition may be administered intravenously to the subject, or may be intended to be administered intravenously to the subject. The composition may be administered once approximately every three weeks, or may be intended to be administered once every three weeks.

[0013] This patent or application file includes at least one drawing made in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows a schematic diagram of the anti-CTLA-4 antibody structure. [Figure 2] Figure 2 shows the expected disulfide bond structure of the anti-CTLA-4 antibody. [Figure 3] Figure 3 shows the expected disulfide bond structure of the anti-PD-1 antibody. [Figure 4] Figure 4 shows the in vivo effects of anti-PD-1, anti-CTLA-4 mAb, or combination therapy on mouse MC38 tumors in human CTLA4 / PD1 double knock-in mice. When the tumors had grown to an average volume of approximately 360 mm³, human CTLA4 / PD1 double knock-in C57BL / 6 mice were subcutaneously inoculated with MC38 cells (1 × 10⁶ cells / mL, 0.1 mL). The mice were randomly divided into seven different groups (n=6 or 7) and administered a total of five intraperitoneal injections (once every three days) of AI-025, ONC-392, AI-061, or PBS (medium control). Mice were monitored daily, and body weight and tumor size were measured once every three days. [Figure 5] Figure 5 shows the MC38 tumor growth curves in individual mice from different groups. Each line represents tumor growth from a single individual mouse. [Figure 6] Figure 6 shows the survival curve of the mouse shown in Figure 5. [Figure 7] Figure 7 shows the pharmaceutical manufacturing process flow. [Figure 8] Figure 8 shows an overview of the clinical trial design. [Figure 9A] Figures 9A to 9C show the tumor response in patients treated with 600 mg of AI-061. Figure 9A shows the overall response over time, Figure 9B shows the percentage change over time, and Figure 9C shows the best percentage change over time. [Figure 9B] Figures 9A to 9C show the tumor response in patients treated with 600 mg of AI-061. Figure 9A shows the overall response over time, Figure 9B shows the percentage change over time, and Figure 9C shows the best percentage change over time. [Figure 9C] Figures 9A to 9C show the tumor response in patients treated with 600 mg of AI-061. Figure 9A shows the overall response over time, Figure 9B shows the percentage change over time, and Figure 9C shows the best percentage change over time. [Figure 10A] Figures 10A to 10C show the tumor response in patients treated with 400 mg of AI-061. Figure 10A shows the overall response over time, Figure 10B shows the percentage change over time, and Figure 10C shows the best percentage change over time. [Figure 10B] Figures 10A to 10C show the tumor response in patients treated with 400 mg of AI-061. Figure 10A shows the overall response over time, Figure 10B shows the percentage change over time, and Figure 10C shows the best percentage change over time. [Figure 10C] Figures 10A to 10C show the tumor response in patients treated with 400 mg of AI-061. Figure 10A shows the overall response over time, Figure 10B shows the percentage change over time, and Figure 10C shows the best percentage change over time. [Figure 11A]Figures 11A - 11C show the tumor response in patients treated with 200 mg of AI - 061. Figure 11A shows the overall response over time, Figure 11B shows the percentage change over time, and Figure 11C shows the best percentage change over time. [Figure 11B] Figures 11A - 11C show the tumor response in patients treated with 200 mg of AI - 061. Figure 11A shows the overall response over time, Figure 11B shows the percentage change over time, and Figure 11C shows the best percentage change over time. [Figure 11C] Figures 11A - 11C show the tumor response in patients treated with 200 mg of AI - 061. Figure 11A shows the overall response over time, Figure 11B shows the percentage change over time, and Figure 11C shows the best percentage change over time. [Figure 12] Figure 12 shows the average serum ONC - 392 concentration in the PK analysis set. [Figure 13] Figure 13 shows the average serum AI - 025 concentration in the PK analysis set.

Mode for Carrying Out the Invention

[0015] Concurrent combination therapy can be administered either by simultaneous administration of each drug from conventional unit-dose individual vials or co-packaged vials, as is the case with the recently approved Ronaprive [REGEN-COV®, a co-packaged combination of two neutralizing antibodies against the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (casiribimab, IgG1κ and imdevimab, IgG1λ)]. Alternatively, it can be supplied as a combination product consisting of two or more drugs in fixed ratios within the same unit-dose vial. Combination-type pharmaceuticals offer many benefits, including reduced risk of error, easier handling, and simpler administration, leading to improved patient compliance. This represents a growing trend in drug development, as evidenced by the successful introduction of PHESGO® (pertuzumab, trastuzumab, and hyaluronidase-zzxf, Roche / Genentech), a subcutaneous combination of two different anti-Her2 antibodies, pertuzumab and trastuzumab, into clinical applications. This combination demonstrated an acceptable safety, pharmacokinetic, and efficacy profile in clinical trials for the treatment of breast cancer and received approval from the U.S. FDA on June 29, 2020.

[0016] Continuing this trend, Bristol Myer Squibb (BMS) developed OPDUALAG®, a fixed-dose combination drug consisting of the anti-PD-1 mAb nivolumab and the anti-LAG-3 mAb relatrimab in a fixed ratio of 3:1. OPDUALAG® was recently approved for melanoma patients based on data from a Phase 3 trial. More recently, Merck initiated a Phase 3 trial of MK-1308A (combination anti-CTLA-4 mAb MK-1308 and anti-PD-1 pembrolizumab) in participants with advanced clear cell renal cell carcinoma (NCT04736706).

[0017] The inventors have gained insight that the combination of the anti-PD-1 antibody AI-025 and the anti-CTLA-4 antibody ONC-392 offers patient benefits in terms of improved safety and efficacy. This is supported by safety and efficacy data of co-administration of ONC-392 with anti-PD-1 pembrolizumab. Briefly, in a dose-escalation study involving 13 patients receiving either 3 mg / kg or 6 mg / kg of ONC-392 in combination with a fixed dose of 200 mg of pembrolizumab, the irAE rates were similar between the two dose cohorts (1 / 7 in the 3 mg / kg cohort and 1 / 6 in the 6 mg / kg cohort). A 30% ORR was obtained among the 10 evaluable patients. The inventors determined that the combination of the two components not only reduces infusion time and avoids errors, but also reduces exposure to inactive components such as PS80. More importantly, in the combination, each of the two antibodies maintains its native structure and divalent binding to its target antigen. Specifically, in this combination product, AI-025 is a non-depleting humanized IgG4 isotype with high affinity target binding and ligand-blocking activity, while ONC-392 is an Fc-enhanced humanized IgG1 isotype. In preclinical and clinical settings, anti-PD-1 has been well demonstrated to function as an antagonist alone, while ONC-392 relies on the depletion of intratumoral Tregs.

[0018] The inventors determined that ONC-392 and AI-025 have similar PK profiles in patients. The PK of ONC-392 is best described by a two-compartment model with primary elimination. Terminal phase half-life (t 1 / 2The estimated half-life (t1 / 2) was 25.7 days. Baseline albumin was identified as a significant covariate for CL; increased albumin levels were associated with decreased CL. Body weight was identified as a significant covariate for the volume term, including central volume (V1) and peripheral volume (V2); weight gain was associated with increases in V1 and V2. No effects of age, sex, race, AST, bilirubin, creatinine clearance, or cancer type on ONC-392 PK were detected. None of these covariates were considered clinically significant. Notably, concurrent chemotherapy with PD-1 was not a significant PK covariate. Preliminary population pharmacokinetic analysis suggests that the PK profile of AI-025 from clinical trials can be characterized by a two-compartment model with primary elimination. The estimated terminal phase half-life (t1 / 2) was 9.0 to 29.2 days.

[0019] The inventors also gained insight into developing AI-061, a combination drug of ONC-392 and AI-025 in a fixed dose (1:1). This was based on the safety and efficacy profiles of ONC-392 (IgG1 anti-CTLA4 mAb) and AI-025 (IgG4 anti-PD-1 mAb) as determined in preclinical and clinical trials.

[0020] ONC-392 is a humanized monoclonal IgG1κ isotype antibody against CTLA-4. The parental clone was selected for its high antitumor efficacy and low autoimmune toxicity. Under low pH, ONC-392 dissociates from CTLA-4, allowing it to escape lysosomal degradation and recycling to the cell surface. In addition, ONC-392 has a modified human IgG1-Fc region that may extend its half-life by promoting binding to FcRn and increases its ADCC activity by reducing its binding to inhibitory FcRIIB. Thus, ONC-392 belongs to a new generation of anti-CTLA-4 antibodies that retain recycling of both CTLA-4 and ONC-392, and therefore exhibits minimal irAEs while possessing potent antitumor activity. Three pieces of evidence support the idea that the pH-sensitive antibody ONC-392 is not only safer than the pH-insensitive ipilimumab but also more effective in Treg depletion and tumor rejection.

[0021] Firstly, by protecting CTLA-4 on the cell surface of Tregs in the tumor microenvironment, ONC-392 maintains the target density for more potent ADCC activity. In addition, ONC-392 is also recycled after internalization, which allows for higher antibody levels in tumors. As a result of these combined effects, ONC-392 is more effective in selective depletion of Tregs in the tumor microenvironment. Secondly, ONC-392 is significantly more potent in inducing rejection of large tumors in human CTLA4 knock-in mice. Thirdly, by avoiding downregulation of CTLA-4 in Tregs outside the tumor microenvironment, ONC-392 protects the physiological function of CTLA-4 in normal tissues. As a result, ONC-392 causes minimal immune-related adverse events (irAEs) in a CTLA-4 knock-in mouse model that completely replicates the irAEs of currently clinically used anti-CTLA-4 antibodies. Accordingly, the combination of ONC-392 and anti-PD-1 does not induce apparent T cell activation in peripheral lymphoid organs.

[0022] PD-1 interacts with its ligands, PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), which inhibit or weaken the host immune response against autologous tissues and tumors. Compared to CTLA-4, PD-1 has a broader tissue expression pattern, with other innate immune cells such as NK cells, monocytes, and macrophages, in addition to activated T lymphocytes and B lymphocytes, showing steady-state or upregulated PD-1 expression upon stimulation.

[0023] The cytoplasmic domain of PD-1 is relatively long, containing 96 amino acids, and includes both an immunoreceptor tyrosine-dependent repression motif (ITIM) containing tyrosine at 223 and an immunoreceptor tyrosine-dependent switch motif containing tyrosine at 228. When stimulated by TCR / CD3 and CD28 in T cells or bound by their ligands PD-L1 or PD-L2, these tyrosine residues are phosphorylated, and these sites are replenished with Src homology-2 domain-containing tyrosine phosphatases-1 and -2 (SHP-1 and SHP-2), which subsequently weaken TCR-mediated T cell activation and proliferation. The involvement of PD-1 can directly inhibit TcR-induced phosphorylation of downstream signaling to the ZAP70 / CD3ζ signalosome and PKCθ. PD-1 blockade is more effective than CTLA-4 blockade in attenuating CD3 / CD28 costimulation-induced changes in T cell transcription profiles, which can be partially explained by recent in vitro studies showing that PD-1-bound SHP-2 directly phosphorylated CD28 demonstrated cell-free systems in fluorescence resonance energy transfer (FRET) assays. In accordance with these in vitro preclinical data, clinically approved anti-PD-1 antibodies, such as anti-PD-L1 antibodies like nivolumab (Opdivo), pembrolizumab (Keytruda), or atezolizumab (Tecentriq), have all demonstrated potent and broad-spectrum cancer immunotherapy efficacy, both clinically as monotherapy and as part of combination therapy with ipilimumab (anti-CTLA-4).

[0024] AI-025 is a humanized IgG4κ isotype monoclonal antibody against human and monkey PD-1. As an IgG4 isotype antibody that weakly binds to Fc receptors such as C1q or complement, it possesses neither ADCC nor CDC activity. The parent molecule of AI-025 is a mouse monoclonal antibody that did not react with mouse PD-1. Therefore, the nonclinical in-vivo efficacy and on-target toxicity / safety of the AI-025 antibody were primarily tested in a human PD1 knock-in mouse tumor-bearing mouse model. The in-vivo antitumor activity of AI-025 was tested in a human PD-1 knock-in mouse model subcutaneously inoculated with the mouse colon adenocarcinoma cell line MC38. The data showed that AI-025 and Opdivo (nivolumab) possessed comparable antitumor activity. In human PD-1 knock-in mice, the antitumor effect was persistent and long-lasting, characterized by immunological memory or immune recall responses to tumor cell rechallenge.

[0025] 1.Definition The terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting. Where used herein and in the appended claims, the singular forms "a," "an," and "the" include plural subjects unless the context otherwise expressly indicates.

[0026] For the purposes of describing numerical ranges in this specification, numbers between each of these ranges that have the same degree of precision are explicitly intended. For example, for the range 6–9, the numbers 7 and 8 are intended in addition to 6 and 9, and for the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0027] 2. Anti-CTLA-4 antibody This specification provides for anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies may be described in U.S. Patent No. 10,618,960, which is incorporated herein by reference. Anti-CTLA-4 antibodies may comprise a complementation-determining region (CDR) 1 comprising the amino acid sequence RASENIYSNLA (SEQ ID NO: 1); a CDR2 comprising the amino acid sequences AATNLQS (SEQ ID NO: 2) (LC1), AATNLQD (SEQ ID NO: 3) (LC2), or AATSLQS (SEQ ID NO: 4) (LC3); and a light chain variable region comprising a CDR3 comprising the amino acid sequence QHLWGTPYT (SEQ ID NO: 5).

[0028] Each light chain variable region containing one of LC1 to LC3 may contain one of the following sequences:

[0029] LC1 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLLYAATNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHLWGTPYTFGGGTKLEIK(Sequence ID 11)

[0030] LC2 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKQGKAPKLLLYAATNLQDGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQHLWGTPYTFGQGTKLEIK(Sequence ID 12)

[0031] LC3 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIYAATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHLWGTPYTFGGGTKVEIK(Sequence ID 13)

[0032] More specifically, each light chain containing one of LC1 to LC3 may contain one of the following amino acid sequences:

[0033] LC1 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLLYAATNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHLWGTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Sequence ID 22)

[0034] LC2 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKQGKAPKLLLYAATNLQDGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQHLWGTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(Sequence ID 23)

[0035] LC3 DIQMTQSPSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKAPKLLIYAATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHLWGTPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (Sequence ID 24).

[0036] The anti-CTLA-4 antibody may contain a heavy chain variable region including CDR1 containing the amino acid sequence GFSLTSYGLS (SEQ ID NO: 6); CDR2 containing the amino acid sequences YIWYDGNTNFHPSLKSR (SEQ ID NO: 7) (HC1), YIWYDGNTNFHSSLKSR (SEQ ID NO: 8) (HC2); or YIWYDGNTNFHSPLKSR (SEQ ID NO: 9) (HC3); and CDR3 containing the amino acid sequence TEGHYYGSNYGYYALDY (SEQ ID NO: 10).

[0037] More specifically, each heavy chain variable region containing one of HC1 to HC3 may contain one of the following amino acid sequences:

[0038] HC1 QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGLSWIRQPPGKGLEWIGYIWYDGNTNFHPSLKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCAKTEGHYYGSNYGYYALDYWGQGTSVTVSS (Sequence ID 14)

[0039] HC2 QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGLSWIRQPPGKGLEWIGYIWYDGNTNFHSSLKSRVTISKDTSKSQVSLKLSSVTAADTAVYYCAKTEGHYYGSNYGYYALDYWGQGTLVTVSS (Sequence ID 15)

[0040] HC3 QVQLQESGPGLVKPSETLSLTCTVSGFSLTSYGLSWIRQPPGKGLEWIGYIWYDGNTNFHSPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAKTEGHYYGSNYGYYALDYWGQGTLVTVSS (Sequence ID 16).

[0041] Anti-CTLA-4 antibodies may contain a heavy chain constant region from a human Ig protein, which may be IgG, IgE, IgM, IgD, IgA, IgY, IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In one example, the constant region is the Fc region from a human IgG1 protein. In another example, the heavy chain constant region has the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 17) Includes.

[0042] The heavy chain constant region may contain one or more mutations. For the sequence described in SEQ ID NO: 17, one or more mutations may be selected from M135Y, S137T, T139E, S181A, E216A, and K217A, and combinations thereof. In one example, the heavy chain constant region of the antibody contains all six mutations. The mutated heavy chain constant region has the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(Sequence ID 18) It may include.

[0043] More specifically, the heavy chain of an anti-CTLA-4 antibody containing the heavy chain variable regions HC1-HC3 may each contain one of the following amino acid sequences:

[0044] HC1 QVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGLSWIRQPPGKGLEWIGYIWYDGNTNFHPSLKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCAKTEGHYYGSNYGYYALDYWG QGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG** (Sequence ID 19)

[0045] HC2 QVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGLSWIRQPPGKGLEWIGYIWYDGNTNFHSSLKSRVTISKDTSKSQVSLKLSSVTAADTAVYYCAKTEGHYYGSNYGYYALDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG** (Sequence ID 20)

[0046] HC3 QVQLQESGPGLVKPSETLSLTTCTVSGFSLTSYGLSWIRQPPGKGLEWIGYIWYDGNTNFHSPLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAKTEGHYYGSNYGYYALDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG** (Sequence ID 21)

[0047] C-terminal lysine (K) may be additionally included in the heavy chain amino acid sequence described in SEQ ID NOs. 19-21, which may increase the expression level. Terminal lysine may be spontaneously cleaved during the production of anti-CTLA-4 antibodies or upon administration of the antibodies.

[0048] PP4637(LC2 / HC3): For example, the anti-CTLA-4 antibody includes a light chain variable region containing CDR1 with the sequence described in SEQ ID NO: 1, CDR2 with the sequence described in SEQ ID NO: 3, and CDR3 with the sequence described in SEQ ID NO: 5. The heavy chain variable region includes CDR1 with the sequence described in SEQ ID NO: 6, CDR2 with the sequence described in SEQ ID NO: 9, and CDR3 with the sequence described in SEQ ID NO: 10. More specifically, the light chain variable region may contain the sequence described in SEQ ID NO: 12, and the heavy chain variable region may contain the sequence described in SEQ ID NO: 16. More specifically, the light chain may contain the sequence described in SEQ ID NO: 23, and the heavy chain may contain the sequence described in SEQ ID NO: 21. This antibody may be called ONC-392. The structure of the anti-CTLA-4 antibody ONC-392 is shown in Figure 1. A schematic diagram of the expected disulfide bond structure of ONC-392 is shown in Figure 2.

[0049] PP4631(LC2 / HC1): In an alternative example, the anti-CTLA-4 antibody includes a light chain variable region containing CDR1 with the sequence described in SEQ ID NO: 1, CDR2 with the sequence described in SEQ ID NO: 3, and CDR3 with the sequence described in SEQ ID NO: 5. The heavy chain variable region includes CDR1 with the sequence described in SEQ ID NO: 6, CDR2 with the sequence described in SEQ ID NO: 7, and CDR3 with the sequence described in SEQ ID NO: 10. More specifically, the light chain variable region may contain the sequence described in SEQ ID NO: 13, and the heavy chain variable region may contain the sequence described in SEQ ID NO: 14. More specifically, the light chain may contain the sequence described in SEQ ID NO: 23, and the heavy chain may contain the sequence described in SEQ ID NO: 19.

[0050] PP4638(LC3 / HC3): In a further example, the anti-CTLA-4 antibody includes a light chain variable region containing CDR1 containing the sequence described in SEQ ID NO: 1, CDR2 containing the sequence described in SEQ ID NO: 4, and CDR3 containing the sequence described in SEQ ID NO: 5. The heavy chain variable region includes CDR1 containing the sequence described in SEQ ID NO: 6, CDR2 containing the sequence described in SEQ ID NO: 9, and CDR3 containing the sequence described in SEQ ID NO: 10. More specifically, the light chain variable region may contain the sequence described in SEQ ID NO: 12, and the heavy chain variable region may contain the sequence described in SEQ ID NO: 16. More specifically, the light chain may contain the sequence described in SEQ ID NO: 24, and the heavy chain may contain the sequence described in SEQ ID NO: 21.

[0051] 3. Anti-PD-1 antibody This specification provides an anti-PD-1 antibody. The anti-PD-1 antibody may be described in U.S. Patent No. 11,345,754. The anti-PD-1 antibody may include a light chain containing a light chain variable region that may include one or more of the following: a complementation-determining region (CDR) 1 containing the amino acid sequence KASQDAGSAVA (SEQ ID NO: 25), a CDR2 containing the amino acid sequence WASTRHT (SEQ ID NO: 26), and a CDR3 containing the amino acid sequence QQYSSYPWT (SEQ ID NO: 27). The light chain variable region may include the following sequences: DIQLTQSPSFLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSYPWTFGGGTKLEIK (Sequence ID 28).

[0052] The light chain may contain a constant region that includes the following sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 29).

[0053] The light chain may contain the following sequence: DIQLTQSPSFLSASVGDRVTITCKASQDAGSAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYSSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 30).

[0054] The anti-PD-1 antibody may contain a heavy chain containing a heavy chain variable region which may contain one or more of the following: CDR1 containing the sequence GFTFSRYD (SEQ ID NO: 31), CDR2 containing the sequence ISGGGRYTYY (SEQ ID NO: 32), and CDR3 containing the sequence PYGNYGMDY (SEQ ID NO: 33). The heavy chain variable region may contain the following sequences: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMSWVRQAPGKGLEWVSTISGGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPYGNYGMDYWGQGTSVTVSSA (Sequence ID 34).

[0055] The heavy chain may contain a constant region that could be the wild-type IgG4 constant region, which includes the following sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 35).

[0056] The IgG4 heavy chain may be a mutant IgG4 heavy chain containing a serine-to-proline change at position 226 of the antibody, which may be present in the hinge region. The mutant IgG4 constant region may contain the following sequence: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 36).

[0057] The heavy chain may contain the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYDMSWVRQAPGKGLEWVSTISGGGRYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSPYGNYGMDYWGQGTS VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 37).

[0058] The anti-PD-1 antibody may include a light chain containing the sequence described in SEQ ID NO: 30 and a heavy chain containing the sequence described in SEQ ID NO: 37. This anti-PD-1 antibody may be referred to herein as AI-025. A schematic diagram of the expected disulfide bond structure of the anti-PD-1 antibody AI-025 is shown in Figure 2.

[0059] The CDR of the heavy chain of mAb21 may be translated to the frame region of the human heavy chain variable region (VH) from IgHV3-23 (NCBI Gene ID: 28442), which may have the highest protein sequence homology to the AI-025 heavy chain V region. The CDR of the light chain of mAb21 may be translated to the frame region or human light chain variable region (VL) from IgKV1-9 (NCBI Gene ID: 28941), which may have the highest protein sequence homology to the AI-025 light chain V region.

[0060] Humanized VH may be conjugated to a human IgG4 heavy chain constant region (CH) (UniProtKB / Swiss-Prot:P01861.1) having a serine-to-proline mutation (S226P) at position 226 in the hinge region, and humanized VL may be conjugated to a human κ light chain constant region (UniProtKB / Swiss-Prot:P01834.2) to generate a fully humanized AI-025 antibody of the IgG4 / κ type. The IgG4 heavy chain may reduce the possibility of in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity against PD-1 positive immune cells such as antigen-active T lymphocytes, and the S226P mutation in the hinge region may prevent the generation of IgG4 half-chains and Fab arm exchange.

[0061] 4. Complex antibody composition This specification provides a composition comprising a combination of at least one anti-CTLA-4 antibody and at least one anti-PD-1 antibody. The composition may include a pharmaceutically acceptable carrier. For example, the anti-CTLA-4 antibody is ONC-392 and the anti-PD-1 antibody is AI-025. The composition comprising the combination of ONC-392 and AI-025 may be called AI-061. AI-061 may contain equal amounts of ONC-392 and AI-025. Due to its dual-targeting nature of CTLA-4 and PD-1, AI-061 may be a potent immune checkpoint inhibitor. The anti-CTLA-4 antibody and the anti-PD-1 antibody may be combined in the same composition, which may include the buffers disclosed herein.

[0062] The composition may include a sterile solution for intravenous infusion. The composition may be a preservative-free aqueous solution and may be suitable for parenteral administration. In one example, the composition independently contains about 5, 10, 20, 30, 40, or 50 mg / mL of anti-CTLA-4 antibody and anti-PD-1 antibody, or amounts in this range. The composition may contain about 5, 10, or 20 mg / mL of anti-CTLA-4 antibody and anti-PD-1 antibody, respectively. The composition may include ratios of anti-CTLA-4 antibody to anti-PD-1 antibody of about 10:1, 5:1, 4:1, 3:1, 2:1, 4:3, 3:2, 1:1, 2:3, 3:4, 1:2, 1:3, 1:4, 1:5, or 1:10. In one example, the composition contains 10 mg / mL of anti-CTLA-4 antibody and anti-PD-1 antibody, respectively. In one example, the composition contains 100 mg each of anti-CTLA-4 antibody and anti-PD-1 antibody. The composition may contain about 5, 10, 15, 20, 25, or 30 mL of aqueous solution of the antibodies. The aqueous solution may be contained in a vial. The volume of the aqueous solution in the vial may be 10 mL. In one example, the anti-CTLA-4 antibody and anti-PD-1 antibody are present at a combined protein concentration of 20 mg / mL.

[0063] A pharmaceutically acceptable carrier may comprise one or more of histidine buffer, acetate buffer, trehalose, PS80, sucrose, and EDTA. The composition may comprise about 5, 10, 15, 20, 25, 30, 35, or 40 mM histidine buffer, or two ranges of these amounts. For example, the composition may comprise 20 mM histidine buffer. The composition may contain about 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, or 10.0% (weight / volume) of α,α-trehalose dihydrate, or amounts within two ranges of these amounts. For example, the composition contains 8.8% (weight / volume) of α,α-trehalose dihydrate. The composition may contain approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.10 (weight / volume) of PS80, or an amount within two ranges of these amounts. For example, the composition contains 0.06% (weight / volume) of PS80. The composition may contain approximately 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, or 0.40 mM of EDTA, which may be EDTA·2Na·2H2O. For example, the composition contains 0.2 mM of EDTA. The composition may have a pH of 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or a pH within these ranges.

[0064] In one example, the composition comprises 20 mM histidine buffer, 8.8% (weight / volume) α,α-trehalose dihydrate, and 0.06% (weight / volume) PS80 at a pH of 6.0. In another example, the composition further comprises 0.2 mM EDTA·2Na·2H2O. The composition may also comprise 20 mM acetate buffer, 9% (weight / volume) sucrose, and 0.02% (weight / volume) PS80, and may have a pH of 5.8.

[0065] Each vial may be filled with a total volume of 10 mL of ONC-392 (approximately 100 mg / vial) and AI-025 (100 mg / vial).

[0066] 5. Dosage regimen Anti-CTLA-4 antibodies and anti-PD-1 antibodies may be administered systemically, possibly by injection or intravenous (IV) administration. One or more doses of anti-CTLA-4 antibodies and anti-PD-1 antibodies may be administered to or intended for administration to the subject. Independently, each dose of each antibody may be approximately 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 6 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg, or two of these amounts. Independently, each dose of each antibody may be approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg, or two of these ranges, for subjects weighing 30 kg or more. Each dose of each antibody may be approximately 50 to 1000 mg for subjects weighing 30 kg or more. The administered doses of anti-CTLA-4 antibody and anti-PD-1 antibody may be in a ratio of anti-CTLA-4 antibody to anti-PD-1 antibody of approximately 10:1, 5:1, 4:1, 3:1, 2:1, 4:3, 3:2, 1:1, 2:3, 3:4, 1:2, 1:3, 1:4, 1:5, or 1:10. In one example, anti-CTLA-4 antibody and anti-PD-1 antibody are combined in a single formulation in a 1:1 ratio, with each antibody present in amounts of 100, 200, or 300 mg (a total of 200, 400, or 600 mg of antibodies).

[0067] If the dose of each anti-CTLA-4 antibody and anti-PD-1 antibody is 100 mg, they may be infused over a period of at least 30 minutes, or this infusion may be intended. If the dose of each anti-CTLA-4 antibody and anti-PD-1 antibody is 200 mg or more, they may be infused over a period of at least 60 minutes, or this infusion may be intended.

[0068] The doses of each anti-CTLA-4 antibody and anti-PD-1 antibody may be independently about 1, 2, 3, 6, 10, 15, or 20 mg / kg, or two ranges of these amounts. The dose may contain 1 to 20 mg / kg of each anti-CTLA-4 antibody and anti-PD-1 antibody. The dose may be 50 to 1000 mg of each anti-CTLA-4 antibody and anti-PD-1 antibody. The subject's body weight may be at least 30 kg.

[0069] The antibody may be administered periodically, with each dose of the aforementioned dosage being administered to the subject. In each administration cycle, the dose may differ from the previous dose. The administration may include dose escalation. In one example, the antibody is administered approximately every 1, 2, 3, 4, 5, or 6 weeks. More specifically, the antibody is administered approximately every 3 weeks. When describing the duration of an administration cycle, "approximately" may mean ±1, 2, or 3 days.

[0070] In one example, the dose of anti-CTLA-4 antibody and anti-PD-1 antibody is 10 mg / kg each. Anti-CTLA-4 antibody and anti-PD-1 antibody may be administered in a regimen that includes 10 mg / kg for two doses, followed by 1-6 mg / kg for long-term administration (i.e., each subsequent dose is 1-6 mg / kg). Long-term administration may include doses of 3 mg / kg or 6 mg / kg. In one example, each dose is administered approximately every 3 weeks. In one example, anti-CTLA-4 antibody and anti-PD-1 antibody are administered approximately every 4 weeks. Administration may be carried out over approximately 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, or 52 weeks, or within this range.

[0071] Body weight was found not to be a significant covariate for ONC-392 clearance. Clinical trials showed that in cancer patients, ONC-392, either as monotherapy or in combination with an anti-PD-1 antibody, was well-tolerated at doses of ≤10 mg / kg as monotherapy or at 6 mg / kg when used in combination with a flat dose of 200 mg of pembrolizumab. Therefore, flat doses of each anti-CTLA-4 antibody and anti-PD-1 antibody may be administered, including 100 mg, 200 mg (approximately 1.5 mg / kg each), 400 mg (approximately 3 mg / kg each), 600 mg (approximately 4.5 mg / kg each), 800 mg (approximately 6.0 mg / kg each), or 1000 mg (approximately 7.5 mg / kg each), or two ranges of these amounts.

[0072] 6.Treatment method This specification provides a method for treating cancer in subjects who may require cancer treatment. The method may include administering to the subject a combination of at least one anti-CTLA-4 antibody and at least one anti-PD-1 antibody as disclosed herein. In one example, the anti-CTLA-4 antibody is ONC-392 and the anti-PD-1 antibody is AI-025. In a further example, the combination of anti-CTLA-4 antibody and anti-PD-1 antibody is AI-061. This specification further provides the use of anti-CTLA-4 antibody / anti-PD-1 antibody combinations in pharmaceutical manufacturing for cancer treatment and combinations for use in cancer treatment.

[0073] The aforementioned cancers include cancers such as bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, head and neck squamous cell carcinoma (HNSCC), endometrial cancer, renal cell carcinoma, thyroid cancer, and skin cancer; cancers including squamous cell carcinoma; hematological malignancies of the lymphatic system including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt lymphoma; and acute and chronic myeloid leukemia and promyelocytic leukemia. These may include myeloid hematological malignancies; mesenchymal tumors including fibrosarcoma and rhabdomyosarcoma; other tumors including melanoma, seminoma, tetratocalcinoma, neuroblastoma and glioma; central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma and teratocalcinoma. It is also intended that cancers caused by abnormalities in apoptosis will be treated by the methods and compositions of the present invention. Such cancers may include, but are not limited to, follicular lymphoma, p53 mutation-associated cancers, hormone-dependent tumors of the breast, prostate and ovaries, and precancerous lesions such as familial adenomatous polyposis, as well as myelodysplastic syndromes. In certain embodiments, malignant or malignant proliferative changes (metaplasia and dysplasia), or hyperproliferative diseases, including those of the ovaries, bladder, esophagus, gastrointestinal region, liver, bile ducts, pharyngeal tonsils, breasts, colon, lungs, skin, pancreas, or uterus, are treated or prevented by the methods and compositions disclosed herein. In other specific embodiments, sarcoma, melanoma, or leukemia are treated or prevented by the methods and compositions of the present invention.

[0074] For example, the cancers mentioned are melanoma, non-small cell lung cancer (NSCLC), HNSCC, ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer (e.g., cholangiocarcinoma), adenoid cystic carcinoma (ACC), pancreatic cancer, or triple-negative breast cancer (TNBC). In further examples, NSCLC is PD-1 resistant. Ovarian cancer may be high-grade serous ovarian cancer, which may be primary peritoneal cancer or fallopian tube cancer. TNBC may be PD-1 resistant. Melanoma may be immuno-oncology (IO) resistant.

[0075] In one example, the cancer is a solid tumor. The solid tumor may be one or more that are histologically and cytologically confirmed. The tumor may be in a subject with progressive locally advanced or metastatic disease. The subject may have shown insufficient response to or intolerance to at least one established standard medical anti-cancer treatment. The cancer diagnosis of the subject may be determined by standard practice, such as the National Comprehensive Cancer Center Network (NCCN) Clinical Practice Guidelines for Oncology. [Examples]

[0076] The present invention has several embodiments, as illustrated by the following non-limiting embodiments.

[0077] Example 1 Anti-CTLA-4 antibody and anti-PD-1 antibody combination The in vivo antitumor activity of AI-061 was tested in human CTLA4 and PD1 double knock-in mice using a subcutaneously inoculated mouse colon adenocarcinoma cell line MC38, which is sensitive to PD-1 and CTLA-4 therapy. This test was performed using either the monotherapy ONC-392 (anti-CTLA-4) or AI-025 (anti-PD-1) as controls for comparison. Table 1 summarizes the mouse tumor models, treatment groups, and schedules.

[0078] In short, human CTLA4 / PD1 double knock-in C57BL / 6 mice (6-8 weeks old) are mixed with MC38 cells (1 × 10⁶). 6 Cells / mL (0.1mL) were subcutaneously inoculated, and the tumor was approximately 360mm. 3 Once the mice had grown to a certain stage, they were randomly divided into four different groups (n=6 or 7) and received either phosphate-buffered saline (PBS, media control) or AI-025 (anti-PD-1), ONC-392 (anti-CTLA-4), and AI-061 (anti-CTLA-4 + anti-PD-1) via intraperitoneal injection once every three days for a total of five doses, all at a dose level of 1 mg / kg body weight. Table 1 Summary of mouse tumor models, treatment groups, and schedules [Table 1]

[0079] Mice were observed daily for survival rate and signs of toxicity (mouse body weight was used as a reference index for indirect determination of drug toxicity), and tumor volume (V) was measured every three days using calipers (length × width). Tumor volume (V) = 0.5a × b 2 , a is the longest diameter (length) of the tumor, and b is the shortest diameter (width) of the tumor.

[0080] Table 2 shows the mean tumor volume at different post-treatment days in each group, and Figures 4 and 5 show the tumor growth curves (mean + SEM) for each group or individual mouse, respectively. As shown in the table and figures below, at the same dose level of 1.0 mg / kg, AI-061 treatment exhibited superior antitumor activity compared to treatment with ONC-392 alone or AI-025 alone. At the end of the study (day 46 post-treatment), 5 out of 7 mice in the AI-061 treatment group were tumor-free, while in the PBS, AI-025, or ONC-392 treatment groups, 0 out of 6, 0 out of 7, or 2 out of 7 mice were tumor-free, respectively. AI-061 treatment appears to be safe with a 100% mouse survival rate (Figure 6). Table 2. Mean tumor volume at different post-treatment days in each group. [Table 2]

[0081] Example 2 Formulation development This example demonstrates the development of formulations of antibody compositions disclosed herein. The development included testing designed to select buffer systems, excipients, surfactants, and antioxidants to stabilize proteins for long-term storage. The formulations were developed to prevent product loss and minimize the degradation of purity and bioactivity due to stresses experienced during manufacturing, storage, distribution, and handling. Different buffer systems, excipients, and surfactants were evaluated.

[0082] First, pH / buffer and excipient / surfactant combination screening tests were conducted to determine the optimal buffer system, excipient, and surfactant strength for pharmaceutical formulations. The evaluated buffer systems included histidine buffer, acetate buffer, and mixtures of these two buffers. The evaluated excipients included sucrose and trehalose·2H₂O. The surfactant and range evaluation included polysorbate 80 (PS80), 0.02%–0.06% (weight / volume (w / v)). The evaluated stress conditions included thermal stress (40°C with incubation of 4 weeks or less), shear stress (3 days of stirring at 300 rpm at 25°C), and freeze-thaw stress (freeze-thaw cycles of 5 or less between room temperature and -70°C). Overall, 20 mM histidine buffer pH 6.0 was selected as the optimal buffer system. For the AI-061 formulation, 8.8% (weight / volume) trehalose·2H2O was selected as a possible excipient, and 0.06% (weight / volume) polysorbate 80 was selected as a possible surfactant strength. However, a significant decrease in PS80 content and a major CEX peak (indicating both protein components of the formulation) were observed in this candidate formulation under heat stress conditions, and further testing was conducted to address this issue.

[0083] Antioxidant screening tests were conducted to evaluate whether the addition of antioxidants could prevent the degradation of PS80 and the decrease in the major CEX peak. The buffer systems re-evaluated included histidine buffer and acetate buffer. The excipients and surfactants re-evaluated were 8.8% (weight / volume) trehalose·2H2O and 0.06% (weight / volume) PS80, respectively. The antioxidants evaluated included 10 mM L-methionine and 0.2 mM EDTA·2Na·2H2O. The stress conditions evaluated included heat stress (25°C and 40°C for 4 weeks or less). Based on the antioxidant screening tests, 0.2 mM EDTA·2Na·2H2O was selected as the optimal antioxidant for the AI-061 formulation.

[0084] Then, formulation confirmation tests were conducted to determine the optimal pharmaceutical formulation. AI-025 protein, with a target concentration of 10 mg / mL, mixed with ONC-392, also with a target concentration of 10 mg / mL, was formulated in 20 mM histidine buffer containing 8.8% (weight / volume) trehalose·2H2O, 0.2 mM EDTA·2Na·2H2O, and 0.06% (weight / volume) PS80 at pH 6.0. Subsequently, the stability of the samples was evaluated under different temperature conditions (40°C for up to 4 weeks, 25°C for up to 3 months, and 2-8°C for up to 3 months), stirring at 100 rpm for up to 3 days at 25°C, and freeze-thaw cycles of up to 5 cycles between room temperature and -70°C. The test results demonstrated pharmaceutical stability and compatibility with the planned container closure system.

[0085] Based on the results of formulation screening tests and confirmation tests, 10 mg / mL of AI-025B-DS and 10 mg / mL of ONC-392 in 20 mM histidine buffer containing 8.8% (weight / volume) trehalose·2H2O, 0.2 mM EDTA·2Na·2H2O, and 0.06% (weight / volume) PS80 at pH 6.0 were selected as the final clinical formulation for AI-061, stored at 2-8°C. The excipients used in the AI-061 formulation and their stabilizing roles are summarized in Table 3. Table 3: Role of excipients in pharmaceutical formulations [Table 3]

[0086] The batch formulations for 1 unit and 22L pharmaceutical (DP) clinical batches are summarized in Table 4 below. Actual batch sizes may vary depending on clinical supply and demand. Table 4. Pharmaceutical batch prescriptions [Table 4]

[0087] Example 3 Product Characterization This example provides characterization of the antibody composition formulations provided herein. AI-061 is a combination drug formulation (DP) containing two active pharmaceutical ingredients: ONC-392 and AI-025B-DS, with a fixed target protein concentration ratio of 1:1 between ONC-392 mAb and AI-025B mAb. Characterization tests were performed to evaluate whether there are molecular interactions (higher-dimensional structures) and biological interactions between ONC-392 and AI-025B in the AI-061 DP. The characterization had three objectives, see Table 5 below. Table 5: Objectives for Characterization of ONC-392, AI-025B-DS, and AI-061 [Table 5]

[0088] For Objective 3, AI-061 was characterized by a panel of methods including thermal stability analysis by differential scanning calorimetry (DSC), higher-order structure determination by ultraviolet circular dichroism (UV CD), particle size and distribution analysis by differential spectral scattering (DLS), cell-based efficacy assays for ONC-392 and AI-025B. By comparing the characterization results of AI-061 with the characterization results of ONC-392 and AI-025B individually, it was possible to evaluate whether there were any interactions that could affect safety or efficacy.

[0089] For objectives 1 and 2, samples of ONC-392 and AI-025B shown in Table 6 were used. Table 6 Information on DS lots used in process development characteristics evaluation tests [Table 6]

[0090] Regarding Objective 3, in addition to the single protein samples in Table 6, samples of non-clinical and clinical batches of AI-061 listed in Table 7 were used. The characterization results are summarized in Table 8. The results showed no significant interaction between ONC-392 and AI-025B in the combined AI-061 at the molecular (higher-order structure) and biological levels. Table 7 Information on the AI-061 lot used in process development characteristics evaluation tests. [Table 7] Table 8 Summary of Characterization Test Results [Table 8-1] [Table 8-2] [Table 8-3]

[0091] Example 4 formulation This example illustrates formulations for the antibody compositions disclosed herein. The ONC-392 drug substance formulation contains 20 mM histidine buffer, pH 6.0, containing 8.8% (weight / volume) α,α-trehalose dihydrate and 0.06% (weight / volume) polysorbate 80, while the AI-025 drug substance (AI-025B-DS) formulation contains 20 mM NaAC-HAC, 9% (weight / volume) sucrose, 0.02% (weight / volume) PS80, pH 5.8. Therefore, the two drug substances must be mixed in the appropriate concentrations and ratios and formulated into a combination buffer (AI-061 formulation buffer).

[0092] The pharmaceutical manufacturing method consists of, before aseptic filling, melting of AI-025 and ONC-392 APIs, dilution preparation, preparation of concentrated EDTA·2Na·2H2O stock solution, pooling and mixing, filtration sterilization, stoppering, capping, visual inspection, bulk packaging, and storage at 2-8°C. The nominal protein concentrations of AI-025B-DS and ONC-392 APIs are 20.0 g / L and 30.0 g / L, respectively, and the protein concentration of the mixed pharmaceutical is 20 mg / mL. The concentration of EDTA·2Na·2H2O in the pharmaceutical is 0.2 mM. The 20 mg / mL pharmaceutical is filtered and sterilized through a 0.22 μm filter and filled into 10 mL glass vials. The target filling volume is 10.73 mL, which allows for a 10 mL / vial yield.

[0093] The pharmaceutical manufacturing process flow is shown in Figure 7. In-process testing is performed at critical steps in the pharmaceutical manufacturing method. Before filtration sterilization, several parameters such as pH, osmotic pressure, total protein concentration, protein concentration ratio, EDTA·2Na·2H2O concentration, bioburden, and endotoxin are tested.

[0094] Example 5 Clinical trials on the efficacy of anti-CTLA-4 / anti-PD-1 antibody combination therapy for cancer treatment This embodiment demonstrates that the anti-CTLA-4 / anti-PD-1 antibody combination therapy disclosed herein effectively treats cancer. The drug is AI-061 disclosed herein, with ONC-392 (anti-CTLA-4 antibody) and AI-025 (anti-PD-1 antibody) present in a 1:1 ratio. The clinical trial was a Phase 1 dose-escalation study. Subjects had to meet the following eligibility criteria: adult patients with a confirmed history of solid tumors; disease progression after systemic treatment; RECIST 1.1 measurable lesions; ECOG 0 / 1; adequate organ function; and lactate dehydrogenase (LDH) ≤ upper limit of normal (ULN).

[0095] A total of 18 subjects were enrolled from July 2023 to February 2024. Three fixed dosing regimens were examined: 200 mg every 3 weeks (Q3W) (n=3); 400 mg every 3 weeks (Q3W) (n=9); 600 mg every 3 weeks (Q3W) (n=6) (Figure 8). The subjects had the following tumor types: adenoid cystic carcinoma (1), cervical cancer (2), cholangiocarcinoma (2), colorectal cancer (1), gastroesophageal adenocarcinoma (1), hepatocellular carcinoma (1), melanoma (1), NSCLC (4), ovarian cancer (1), pancreatic cancer (2), and renal cell carcinoma (2). No dose-limiting toxicities were observed. Patient characteristics are shown in the table below. Table 9 [Table 9]

[0096] As of the data cut-off date of March 1, 2024, there were 3 Grade 11 treatment-related adverse events (38%) in 7 subjects. There were no Grade 4 or Grade 5 adverse events (AEs) related to the investigational drug. A summary of the safety data is shown in the table below. Table 10 [Table 10]

[0097] All surviving subjects underwent at least one assessment of tumor response. As of the data cut-off date of April 22, 2024, there were 2 partial responses (PR) (33%) in the 600 mg group (Table 11 and Figures 9A-9C) and 2 partial responses (PR) (22%) in the 400 mg group (Table 12 and Figures 10A-10C). There was no response in the 200 mg group (Table 13 and Figure 11). Table 11 Summary of the 600 mg cohort [Table 11] Table 12 Summary of the 400 mg cohort [Table 12] Table 13 Summary of the 200 mg cohort [Table 13]

[0098] Pharmacokinetic (PK) analyses of ONC-392 (Table 14 and Figure 12) and AI-025 (Table 15 and Figure 13) showed predictable patterns for stable monoclonal antibodies. The similarity of these PK profiles supports their co-administration. Table 14 Summary of Key Pharmacokinetic Parameters of ONC-392 by Cohort and Period in the PK Analysis Set [Table 14] Table 15 Summary of key pharmacokinetic parameters of AI-025 by cohort and time period in the PK analysis set. [Table 15]

[0099] Therefore, the results of the clinical trials indicate that the combination therapy using the anti-CTLA-4 antibody and anti-PD-1 antibody disclosed herein is effective against cancer.

Claims

1. A composition comprising an anti-CTLA-4 antibody and an anti-PD-1 antibody, (a) The anti-CTLA-4 antibody comprises (i) a light chain variable region comprising a complementarity-determining region (CDR) 1 comprising the sequence described in SEQ ID NO: 1, a CDR 2 comprising the sequence described in SEQ ID NO: 3, and a CDR 3 comprising the sequence described in SEQ ID NO: 5; and (ii) a heavy chain variable region comprising a CDR 1 comprising the sequence described in SEQ ID NO: 6, a CDR 2 comprising the sequence described in SEQ ID NO: 9, and a CDR 3 comprising the sequence described in SEQ ID NO: 10; and, (b) The anti-PD-1 antibody comprises (i) a light chain variable region including CDR1 containing the sequence described in SEQ ID NO: 25, CDR2 containing the sequence described in SEQ ID NO: 26, and CDR3 containing the sequence described in SEQ ID NO: 27; and (ii) a heavy chain variable region including CDR1 containing the sequence described in SEQ ID NO: 31, CDR2 containing the sequence described in SEQ ID NO: 32, and CDR3 containing the sequence described in SEQ ID NO:

33. composition.

2. The composition according to claim 1, wherein the anti-CTLA-4 antibody comprises a light chain variable region containing the sequence described in SEQ ID NO: 12 and a heavy chain variable region containing the sequence described in SEQ ID NO:

16.

3. The composition according to claim 2, wherein the anti-CTLA-4 antibody comprises a light chain containing the sequence described in SEQ ID NO: 23 and a heavy chain containing the sequence described in SEQ ID NO:

21.

4. The composition according to any one of claims 1 to 3, wherein the anti-PD-1 antibody comprises a light chain variable region containing the sequence described in SEQ ID NO: 28 and a heavy chain variable region containing the sequence described in SEQ ID NO:

34.

5. The composition according to claim 4, wherein the anti-PD-1 antibody comprises a heavy chain containing IgG4 having the sequence described in SEQ ID NO: 35 or SEQ ID NO:

36.

6. The composition according to claim 5, wherein the anti-PD-1 antibody comprises a light chain containing the sequence described in SEQ ID NO: 30 and a heavy chain containing the sequence described in SEQ ID NO:

37.

7. The composition according to claim 1, wherein the anti-CTLA-4 antibody comprises a light chain containing the sequence described in SEQ ID NO: 23 and a heavy chain containing the sequence described in SEQ ID NO: 21; and the anti-PD-1 antibody comprises a light chain containing the sequence described in SEQ ID NO: 30 and a heavy chain containing the sequence described in SEQ ID NO:

37.

8. The composition according to claim 1, comprising 1 to 50 mg / mL each of the anti-CTLA-4 antibody and the anti-PD-1 antibody.

9. The composition according to claim 8, comprising 10 mg / mL each of the anti-CTLA-4 antibody and the anti-PD-1 antibody.

10. The composition according to claim 1, comprising a pharmaceutically acceptable carrier.

11. The composition according to claim 10, comprising one or more of histidine buffer, α,α-trehalose dihydrate, polysorbate 80 (PS80), and EDTA.

12. The composition according to claim 11, comprising 5 to 40 mM histidine buffer, 7.0% to 10.0% (weight / volume) of α,α-trehalose dihydrate, and 0.01 to 0.10 (weight / volume) of PS80, wherein the pH of the composition is 5.5 to 6.

5.

13. The composition comprises 20 mM histidine buffer, 8.8% (weight / volume) α,α-trehalose dihydrate, 0.06% (weight / volume) PS80, and 0.2 mM EDTA-2Na-2H ２ The composition according to claim 12, comprising O, wherein the pH of the composition is 6.

0.

14. A method for treating cancer in a subject requiring cancer treatment, comprising administering the composition described in claim 1 to the subject.

15. The method according to claim 14, wherein the cancer is a tumor.

16. The method according to claim 14, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), and triple-negative breast cancer (TNBC).

17. The method according to claim 16, wherein the ovarian cancer is a high-grade serous ovarian cancer.

18. The method according to claim 17, wherein the high-grade serous ovarian cancer is primary peritoneal cancer or fallopian tube cancer.

19. The method according to claim 14, wherein the cancer is PD-1 resistant.

20. The method according to claim 14, comprising administering to the subject 10 mg / kg or less of each of the anti-CTLA-4 antibody and the anti-PD-1 antibody.

21. The method according to claim 14, comprising administering the composition intravenously.

22. The method according to claim 14, wherein the composition is administered once every three weeks.

23. A composition comprising an anti-CTLA-4 antibody and an anti-PD-1 antibody, (a) The anti-CTLA-4 antibody comprises a light chain containing the sequence described in SEQ ID NO: 23 and a heavy chain containing the sequence described in SEQ ID NO: 21; (b) The anti-PD-1 antibody comprises a light chain containing the sequence described in SEQ ID NO: 30 and a heavy chain containing the sequence described in SEQ ID NO: 37; (c) The composition comprises 20 mM histidine buffer, 8.8% (weight / volume) α,α-trehalose dihydrate, 0.06% (weight / volume) PS80, and 0.2 mM EDTA-2Na-2H ２ Including O; and (d) The pH of the composition is 6.

0. composition.

24. The composition according to claim 23, comprising 10 mg / mL each of the anti-CTLA-4 antibody and the anti-PD-1 antibody.

25. A method for treating cancer in a subject requiring cancer treatment, comprising administering the composition described in claim 23 to the subject.

26. The method according to claim 25, wherein the cancer is a tumor.

27. The method according to claim 25, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), pancreatic cancer, and triple-negative breast cancer (TNBC).

28. The method according to claim 25, wherein the composition is administered intravenously.

29. The method according to claim 25, comprising administering 10 mg / kg each of the anti-CTLA-4 antibody and the anti-PD-1 antibody to the subject.

30. The method according to claim 25, wherein the composition is administered once every three weeks.

31. Use of the composition according to any one of claims 1 to 13, 23, and 24 in the manufacture of a pharmaceutical product for cancer treatment.

32. The use according to claim 31, wherein the cancer is a tumor.

33. The use according to claim 31 or 32, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), pancreatic cancer, and triple-negative breast cancer (TNBC).

34. The use of the pharmaceutical product according to any one of claims 31 to 33, wherein the pharmaceutical product is intended to be administered intravenously.

35. The use of the pharmaceutical product according to any one of claims 31 to 34, wherein the pharmaceutical product is intended to be administered once every three weeks.

36. A composition for cancer treatment according to any one of claims 1 to 13, 23, and 24.

37. The composition according to claim 36, wherein the cancer is a tumor.

38. The composition according to claim 36 or claim 37, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), ovarian cancer, endometrial cancer, cervical cancer, renal cell carcinoma, bladder cancer, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, colorectal cancer, anal cancer, hepatocellular carcinoma, bile duct cancer, adenoid cystic carcinoma (ACC), pancreatic cancer, and triple-negative breast cancer (TNBC).

39. A composition according to any one of claims 36 to 38, intended for intravenous administration.

40. A composition according to any one of claims 36 to 39, intended to be administered once every three weeks.

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