Anti-CTLA4 antibodies for treating non-small cell lung cancer

Abstract

The present invention relates to an anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer in a subject in need thereof, wherein the non-small cell lung cancer has a squamous histology. The invention further provides a method of treating non-small cell lung cancer in a subject in need thereof, wherein the method comprises administering the anti-CTLA4 antibody or fragment thereof to the subject, and wherein the non-small cell lung cancer has a squamous histology. The invention also provides a composition comprising the anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer in a subject in need thereof, wherein the non-small cell lung cancer has a squamous histology.

Description

[0001] ANTI-CTLA4 ANTIBODIES FOR TREATING NON-SMALL CELL LUNG CANCER

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for treating non-small cell lung cancer in a subject in need thereof by administering an anti-CTLA antibody or a fragment thereof. The invention further provides a composition comprising the anti-CTLA4 antibody or fragment thereof for use in treating non-small cell lung cancer in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Lung cancer is the world's leading cancer indication accounting for the highest mortality rates among both men and women. Among lung cancer, non-small cell lung cancer (NSCLC) is the most frequent cancer type. The most common types of NSCLC are squamous cell carcinoma (also referred to as squamous cell carcinoma of the lung), large-cell carcinoma, and adenocarcinoma. While treatment options for NSCLC such as surgery, radiation therapy, and chemotherapy exist, there is still a need for new and more effective treatment options. Recently, immune checkpoint inhibitor cancer immunotherapy therapy has attracted great attention from both the scientific and clinical communities.

[0006] Immune checkpoints play a pivotal role in the regulation of the immune response in tumor microenvironments. Immune checkpoints act as gatekeepers of the immune system modulating the nature, magnitude, and duration of the immune response and maintaining selftolerance. One of the key inhibitory immune checkpoints is Cytotoxic T lymphocyte antigen- 4 (CTLA4), also known as CD 152 (cluster of differentiation 152). The interaction between the B7.1 (CD80) (Freeman et al., J Immunol 1989, 143(8): 2714-22) and B7.2 (CD86)

[0007] [P2025W001] / / 241356WO (Freeman et al., Science 1993, 262(5135): 909-11; Hathcock et al., Science 1993, 262(5135): 905-7; Wu et al., J Exp Med 1993, 178(5): 1789-93) ligands of antigen presenting cells and the CD28 and CTLA4 receptors (Leach et al., Science 1996, 271(5256): 1734-6; Linsley et al, J Exp Med 1991, 174(3): 561-9; Linsley et al., Proc Natl Acad Sci U S A 1990, 87(13): 5031- 5) of T cells governs the activation or downregulation of T cells. CTLA4 is recognized as a key regulator of the adaptive immune response, having a central role in the maintenance of peripheral tolerance and in shaping the repertoire of emerging T cell responses and, is therefore a therapeutic target for the treatment of cancer and inflammation.

[0008] Anti-CTLA4 monoclonal antibodies (mAbs) such as the approved antibody, ipilimumab (YERVOY® by Bristol Myers Squibb), have demonstrated strong and broad cancer immunotherapeutic effects (CITE) in a variety of preclinical models and ipilimumab is used clinically both as monotherapy and as part of combination therapy with nivolumab (anti-PD-1 antibody, OPDIVO® by Bristol Myers Squibb). However, anti-CTLA4 antibody monotherapy, in the case of ipilimumab, has more immunotherapy -related adverse effects (irAEs) than drugs targeting the checkpoint inhibitor PD-1, such as anti-PD-(L)l therapy. The strong irAEs further limit the doses tolerated by cancer patients. Nevertheless, CTLA4 remains a valid and attractive immunotherapy target, however, the less favorable safety profile significantly limits its clinical usage.

[0009] The molecular basis underlying irAEs and cancer immunotherapeutic effects (CITE) of anti- CTLA4 antibodies is traditionally viewed as antagonizing the endogenous function of CTLA4. In both mice and humans, genetic inactivation of CTLA4 caused severe autoimmune diseases. Therefore, an effective antagonist of CTLA4 is expected to likely induce autoimmune diseases. If inactivation of CTLA4 is necessary, then irAEs are expected to be a necessary price for cancer immunity.

[0010] Therefore, there is a desire to improve the therapeutic potential of anti-CTLA4 antibodies by increasing CITE efficacy while reducing the associated irAEs. In the past, different therapeutic immune check inhibitors were combined to enhance anti-tumor activity,

[0011] [P2025W001] / / 241356WO particularly against poorly immunogenic tumors. However, this approach is associated with the risk of further increasing the autoimmune side effects further highlighting the need to selectively modulate cancer immunity without enhancing autoimmunity.

[0012] Besides autoimmunity -related toxicity of immune checkpoint cancer immunotherapy, primary or acquired resistance of patients to treatment with immune checkpoint inhibitors is another central challenge in the field of cancer immunotherapy. For patients with primary or acquired resistance to treatment with immune checkpoint inhibitors, further cancer therapy options are limited with most being palliative. A clinical trial involving NSCLC patients with acquired resistance to anti-PD-l / anti-PD-Ll antibody treatment demonstrated that further treatment with a combination of an anti-CTLA4 antibody (tremelimumab, Imjudo® by AstraZeneca) and an anti-PD-Ll antibody (durvalumab, Imfinzi® by AstraZeneca) showed only minimal treatment success, i.e., an overall response rate of 4%.

[0013] Furthermore, the applicability of cancer immunotherapy for NSCLC is limited for patients who lack oncogenic driver mutations that can be targeted by cancer immunotherapeutic approaches.

[0014] Also, as shown in Table 1 below, late-stage clinical trials did not provide a satisfactory overall survival (OS) or tumor objective response rate (ORR), demonstrating the unmet medical needs of treating NSCLC with squamous histology (squamous NSCLS or sqNSCLC).

[0015] Table 1 Data of late-stage clinical trials of patients affected with sqNSCLC

[0016] [P2025W001] / / 241356WO

[0017] * Docetaxel (D)

[0018] ** Sacituzumab govitecan (SG)

[0019] *** Cabozantinib and Atezolizumab (CA)

[0020] NA = not analysed

[0021] Therefore, there is an unmet medical need for improved cancer immunotherapeutic methods and new and improved cancer therapies targeting NSCLC and addressing the challenges posed by, e.g., autoimmune side effects, resistance to treatment with immune checkpoint inhibitors, and lack of targetable oncogenic driver mutations.

[0022] SUMMARY OF THE INVENTION

[0023] Against the aforementioned background, it is therefore an object of the present invention to provide safe and effective immunotherapeutic treatment options for subjects afflicted with NSCLC. It is a further object of the present invention to provide effective therapeutic means for the treatment of NSCLC with reduced autoimmune side effects. It is also an object of the present invention to provide improved immunotherapeutic treatment options for subjects afflicted with NSCLC and resistance to treatment with immune checkpoint inhibitors and / or chemotherapy agents, particularly to patients afflicted with NSCLC who are resistant to treatment with anti-PD-l / PL-Ll inhibitors and / or platinum-based chemotherapy. It is a further object of the present invention to provide improved cancer treatment methods for subjects afflicted with NSCLC who lack targetable oncogenic driver mutations. It is a further object of the present invention to provide safe and effective treatment options for patients afflicted with NSCLC who are resistant to treatment with anti-PD-l / PD-Ll inhibitors and / or platinum-based chemotherapy and who are negative for targetable oncogenic driver mutations.

[0024] [P2025W001] / / 241356WO These objects are achieved by the invention set forth in the claims and embodiments explained in more detail below.

[0025] The invention provides an anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology.

[0026] Challenges of cancer immunotherapy, and in particular immunotherapy of NSCLC with immune checkpoint inhibitors, are the undesired side effects associated with the treatment, such as immune-related adverse events and the resistance of patients to the treatment. In the research underlying the invention, the applicants surprisingly found that a therapy with an anti-CTLA4 antibody provided an unexpected beneficial effect in the treatment of NSCLC with squamous histology, while in parallel providing a safe treatment.

[0027] This surprising effect results in longer overall survival, longer progression-free survival, longer freedom from disease progression (stable disease state), reduction of tumor-related symptoms, and / or reduction of need for pain medications during and / or following the anti- CTLA4 antibody therapy. The effect can be reflected in an improved quality of life, such as mobility, strength of appetite, and / or psychological status.

[0028] The invention further provides a method of treating NSCLC in a subject in need thereof, wherein the method comprises administering an anti-CTLA4 antibody or fragment thereof to the subject, and wherein the NSCLC has a squamous histology.

[0029] The invention further concerns a composition comprising an anti-CTLA4 antibody or fragment thereof for use in a method of treating NSCLC in a subject in need thereof, wherein the NSCLC has a squamous histology.

[0030] Advantageous embodiments of the invention are indicated in the dependent claims and in the following.

[0031] [P2025W001] / / 241356WO DETAILED DESCRIPTION

[0032] Although certain embodiments of the present invention are described in detail below, it is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0033] In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0034] The above objects are achieved by the following embodiments in accordance with the invention:

[0035] 1. An anti-CTLA4 antibody or fragment thereof for use in a method of treating nonsmall cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology.

[0036] 2. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 1, wherein the anti-CTLA4 antibody or fragment thereof comprises:

[0037] [P2025W001] / / 241356WO a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1, (2) a complementarity-determining region 2 (HCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2, and (3) a complementarity-determining region 3 (HCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain variable region comprising (1) a complementarity-determining region 1 (LCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4, (2) a complementarity-determining region 2 (LCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 5, and (3) a complementarity-determining region 3 (LCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 6. The anti-CTLA4 antibody or fragment thereof for use according to embodiments 1 or 2, wherein the anti-CTLA4 antibody is administered as monotherapy. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-3, wherein the anti-CTLA4 antibody or fragment thereof dosage administered ranges from about 0.1 mg / kg to about 45 mg / kg body weight, optionally from about 1 mg / kg to about 30 mg / kg body weight, optionally from about 2 mg / kg to about 20 mg / kg body weight. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-4, wherein the anti-CTLA4 antibody or fragment thereof dosage administered is independently selected from about 1 mg / kg body weight, about 3 mg / kg body weight, about 6 mg / kg body weight, about 10 mg / kg body weight, about 12 mg / kg body weight, about 15 mg / kg body weight, and about 20 mg / kg body weight, optionally, wherein the anti-CTLA4 antibody or fragment thereof dosage

[0038] [P2025W001] / / 241356WO administered is independently selected from about 3 mg / kg body weight, about 6 mg / kg body weight, and about 10 mg / kg body weight. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-5, wherein a treatment cycle is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-6, wherein each treatment cycle has about 18 to about 28 days, optionally about 18 to about 24 days, optionally about 21 days. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-7, wherein the anti-CTLA4 antibody or fragment thereof is administered about every 4 weeks, optionally about every 3 weeks. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-8, wherein the anti-CTLA4 antibody or fragment thereof is administered for 17 treatment cycles or up to 1 year. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-9, wherein the subject is treated with 1 dosage of the anti-CTLA4 antibody or fragment thereof per treatment cycle. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-10, wherein the subject is treated with at least 1 dosage of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof, optionally wherein the subject is treated with at least 2 dosages of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof.

[0039] [P2025W001] / / 241356WO The anti-CTLA4 antibody or fragment thereof for use according to embodiments 1-11, wherein the subject is treated with 1 dosage of about 3, about 6 or about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle, with the provision that the treatment of the subject comprises at most 2 treatment cycles with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 12, comprising administering a first dose of about 10 mg / kg body weight, a second dose of about 10 mg / kg body weight, and one or more subsequent doses of about 1-6 mg / kg body weight, optionally wherein each subsequent dose is about 6 mg / kg body weight. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-10, wherein the subject is treated with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-14, wherein the anti-CTLA4 antibody or fragment thereof is administered on the first day of each treatment cycle. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-15, wherein the anti-CTLA4 antibody or fragment thereof is administered intravenously. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 16, wherein the anti-CTLA4 antibody or fragment thereof is administered via an intravenous injection or an intravenous infusion, optionally an intravenous infusion.

[0040] [P2025W001] / / 241356WO The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-17, wherein the subject is a human. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-18 wherein the anti-CTLA4 antibody or fragment thereof is formulated with one or more pharmaceutically acceptable carriers, diluents and / or excipients. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-19, wherein the subject has not previously been treated for cancer, optionally wherein the subject has previously not been treated for NSCLC with squamous histology. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-19, wherein the subject has been previously treated for cancer, optionally wherein the subject had at least one previous immune checkpoint inhibitor treatment and / or at least one previous chemotherapy treatment. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 21, wherein the at least one previous immune checkpoint inhibitor treatment comprises or is a PD-1 and / or PD-L1 immune checkpoint inhibitor-based treatment. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 21 or 22, wherein the at least one previous chemotherapy treatment comprises or is a platinum-comprising chemotherapy. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 23, wherein the platinum-containing chemotherapy comprises or is selected from

[0041] [P2025W001] / / 241356WO satraplatin, cisplatin, carboplatin, nedaplatin, lobaplatin, oxaliplatin or combinations thereof. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-24, wherein the overall survival in said subject is increased compared to a monotherapy with a chemotherapy agent. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-25, wherein the median progression-free survival in said subject is increased compared to a monotherapy with a chemotherapy agent. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-26, wherein the NSCLC with squamous histology has locally advanced or metastasized. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-27, wherein the NSCLC with squamous histology is negative for targetable NSCLC driver mutations. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 28, wherein the NSCLC with squamous histology is negative for EGFR and / or ALK mutations. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-27, wherein the NSCLC with squamous histology is positive for KRAS mutations.

[0042] [P2025W001] / / 241356WO The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-30, wherein the wherein the anti-CTLA4 antibody or fragment thereof comprises: a) a heavy chain variable domain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7; and b) a light chain variable domain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-31, wherein the anti-CTLA4 antibody or fragment thereof comprises a constant region optionally derived from a human antibody, optionally the constant region is selected from the constant region of human IgGl, IgG2, IgG3 or IgG4. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 32, wherein the constant region is human IgGl constant region. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 33, wherein the anti-CTLA4 antibody or fragment thereof comprises an IgGl Fc, optionally comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-34, wherein the anti-CTLA4 antibody comprises:

[0043] [P2025W001] / / 241356WO a) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-35, wherein the anti-CTLA4 antibody or fragment thereof is capable of binding to human CTLA4. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-36, wherein the anti-CTLA antibody or fragment thereof is a humanized anti-CTLA4 antibody or fragment thereof. The anti-CTLA4 antibody or fragment thereof for use according to any one of embodiments 1-31, 36, or 37, wherein the anti-CTLA antibody fragment is an antigenbinding fragment or a variant thereof. The anti-CTLA4 antibody or fragment thereof for use according to embodiment 38, wherein the antigen binding fragment or variant thereof is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a rlgG, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof. A method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the method comprises administering an anti-CTLA4 antibody or fragment thereof to the subject, and wherein the NSCLC has a squamous histology.

[0044] [P2025W001] / / 241356WO 41. A composition comprising an anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology.

[0045] 42. The composition according to embodiment 41, wherein the composition is a pharmaceutical composition.

[0046] 43. The pharmaceutical composition according to embodiment 42, comprising one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0047] 44. The pharmaceutical composition according to any one of embodiments 42-43, wherein the anti-CTLA-4 antibody has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL, from a formulation containing 30-60 mg / mL anti-CTLA-4 antibody or fragment thereof, 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0.

[0048] Definitions

[0049] The terms indicated for explanation of the invention have the following meaning, unless otherwise indicated in the description or the embodiments. Additional definitions are set forth throughout the detailed description.

[0050] Terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the embodiments) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0051] The terms “about” or “approximately” as used herein denotes a range of ±10% of a reference value. For example, “about 10” defines a range of 9 to 11. In general, those skilled in the art,

[0052] [P2025W001] / / 241356WO familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context.

[0053] The term “plurality” refers to the state of being plural.

[0054] Unless expressly specified otherwise, the term “comprising” is used in the context of the present disclosure to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as specific embodiments of the present invention that each time the term “comprising” is used, this shall also encompass the possibility of no further members being present, i.e., for the purpose of this embodiment “comprising” can be understood as having the meaning of “consisting of’.

[0055] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. K51bl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0056] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012).

[0057] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0058] [P2025W001] / / 241356WO Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0059] The terms “polynucleotide” and “nucleic acid” can be used interchangeably herein to refer to polymers of nucleotides. The term “polynucleotide” comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a polynucleotide is DNA. In some embodiments, a polynucleotide is RNA. A polynucleotide may be present as a mixture of DNA and RNA. A polynucleotide may be further present as a single-stranded or doublestranded and linear or covalently circularly closed molecule. A polynucleotide can be isolated. The term “isolated polynucleotide “ means, according to the present invention, that the polynucleotide (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0060] The term “RNA” relates to a nucleic acid molecule which includes ribonucleotide residues. In some embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2'-position of a P- D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or

[0061] [P2025W001] / / 241356WO deoxynucleotides. For the present invention, these altered / modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered / modified nucleotides (i.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains “a majority of ribonucleotide residues” if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof). The term “RNA” further includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, “RNA” refers to mRNA. RNA as described herein may comprise in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0062] The term “mRNA” refers to messenger RNA that comprises a coding sequence encoding a polypeptide. An mRNA may further comprise non-coding sequences such as a 5 ’cap, a 5’UTR, a 3’UTR, a Kozak sequence, an FI element or a poly(A) tail or any combination thereof. According to the present disclosure, the term “mRNA” means “messenger-RNA” and includes a “transcript” which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide. mRNA is single-stranded but may contain self- complementary sequences that allow parts of the mRNA to fold and pair with itself to form

[0063] [P2025W001] / / 241356WO double helices. According to the present disclosure, “dsRNA” means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0064] Herein, the term “DNA” relates to a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues. In some embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, “deoxyribonucleotide” refers to a nucleotide which lacks a hydroxyl group at the 2’-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides.

[0065] The term “nucleoside” relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2'- deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG. A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups,

[0066] [P2025W001] / / 241356WO e.g., one or more C1-4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5- alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-Ci-4 alkyl-guanine, N6-CI-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(l)-Ci-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(l)- methyl-uracil.

[0067] The term “in vitro transcription” or “IVT” means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).

[0068] As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA- dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or a single-stranded RNA (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent. In some embodiments, a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA. As is understood in the art, the phrase “nucleic acid encoding a peptide or protein” means that the nucleic acid, if present in the appropriate environment, for example within a cell and / or in a cell-free translation system, can direct the assembly of amino acids to produce the peptide or protein via a process of translation.

[0069] [P2025W001] / / 241356WO The terms “identity” or “sequence identity” refer to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. “Sequence identity” between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences. The terms “% identical”, “% identity” or similar terms refer to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sei. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLASTP, BLASTN and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). For example, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at the website blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST _SPEC=blast2seq&LINK_LOC=align2seq). The algorithm parameters used for BLASTN algorithm on the NCBI website may include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to O; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. The algorithm parameters used for BLASTP algorithm on the NCBI website may include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to O; (iv) Matrixset to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared

[0070] [P2025W001] / / 241356WO correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100. In some embodiments, the degree of identity is given for a region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid or amino acid sequence consists of 200 nucleotides or amino acids, the degree of identity is given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides or amino acids, in some embodiments in continuous nucleotides or amino acids. In some embodiments, the degree of identity is given for the entire length of the reference sequence. Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence. One important property includes an immunogenic property, in particular when administered to a subject. In some embodiments, a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to the given sequence.

[0071] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids.

[0072] The term “recombinant” when used in the context of a polynucleotide means a polynucleotide having nucleotide sequences that are not naturally joined together and can be made by artificially combining two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Recombinant polynucleotides include vectors comprising an amplified or assembled polynucleotide, which can be used to transform or transfect a suitable host cell. A host cell that comprises the recombinant polynucleotide is referred to as a “recombinant host

[0073] [P2025W001] / / 241356WO cell.” The polynucleotide is then expressed in the recombinant host cell to produce a “recombinant polypeptide.” A recombinant polynucleotide can also comprise a non-coding function.

[0074] The term “full length” with respect to a given polypeptide means the form of the polypeptide naturally translated from the coding DNA sequence, beginning with the ATG start codon, which encodes the first methionine in the amino acid sequence, and ending at the TGA, TAG, or TTA stop codon, or whichever stop codon employed by the organism.

[0075] The term “gene” refers to a DNA sequence in a chromosome that codes for a protein. In some embodiments, a gene includes coding sequence (i.e., sequence that encodes a particular protein); in some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type- specific expression, inducible expression, etc.).

[0076] The term “carrier” refers to a component which may be natural, synthetic, organic, inorganic in which the active ingredients of the invention are combined in order to facilitate, enhance or enable administration of the anti-CTLA4 antibody or fragment thereof. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to the subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, trehalose dihydrate, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers. Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0077] [P2025W001] / / 241356WO The term “excipient” is a substance which may be present in a formulation of the anti-CTLA4 antibody or fragment thereof but is not an active ingredient. Examples of excipients include, without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0078] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means “carriers” and / or “excipients” such as solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. In certain embodiments, the pharmaceutically acceptable carrier or excipient is not naturally occurring. The term “pharmaceutically acceptable” refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0079] The term “diluent” relates to a diluting and / or thinning agent. Moreover, the term “diluent” includes any one or more of fluid, liquid or solid suspension and / or mixing media. Nonlimiting examples of suitable diluents include ethanol, glycerol, dextrose, and water.

[0080] The term “subject” relates to a human of either gender (a male or a female). The subject may be of any age. In some embodiments, the subject is female. In some embodiments, the subject is male. In some embodiments, the subject is a subject having cancer, in particular a female subject having cancer and / or a male subject having cancer. In some embodiments, the subject is a subject having NSCLC, in particular a female subject having NSCLC and / or a male subject having NSCLC. In some embodiments, the subject is a subject having NSCLC with squamous histology, in particular a female subject having NSCLC with squamous histology and / or a male subject having NSCLC with squamous histology. The subject can be a patient.

[0081] The term “treating” when used in the context of a disease or disease condition means ameliorating, improving or remedying a disease, disorder, or symptom of a disease or condition associated with the disease, or can mean completely or partially stopping, on a

[0082] [P2025W001] / / 241356WO molecular level, the biochemical basis of the disease. It describes an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a disease.

[0083] The term “pharmaceutical composition” relates to a composition comprising an active agent or a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and / or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject. For example, a pharmaceutical composition can comprise the CTLA4-antibody disclosed herein.

[0084] The term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. The term “tumor” as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size and includes primary tumors and secondary neoplasms.

[0085] The term “oncogenic driver mutation(s)” or “driver mutations” as used herein refers to mutations that are responsible for both the initiation and maintenance of a cancer. These mutations are often found in genes that encode for signaling proteins that are critical for maintaining normal cellular proliferation and survival.

[0086] The term “NSCLC driver mutation” as used herein refers to oncogenic driver mutations of non-small cell lung cancer (NSCLC). Non-limiting examples of NSCLC driver mutations are listed in Luo et al., Transl Respir Med. 2013, 1 (1 ): 6, and are e.g., epidermal growth factor receptor (EGFR), MET, anaplastic lymphoma kinase (ALK), c-ROS oncogene 1, v-raf murine sarcoma viral oncogene homolog B, neurotrophic receptor tyrosine kinase, human epidermal growth factor 2, neuregulin-1, and rearranged during transfection, in particular EGFR gene mutations and ALK gene mutations.

[0087] [P2025W001] / / 241356WO The terms “non-small cell lung cancer (NSCLC)” or “non-small cell lung carcinoma” are used interchangeably herein. The most common types of NSCLC are squamous cell carcinoma, large-cell carcinoma, and adenocarcinoma. In the context of the present disclosure, the terms “NSCLC with squamous histology”, “squamous cell carcinoma of the lung”, “squamous cell carcinoma” as a type of NSCLC, and “squamous NSCLC” are used interchangeably herein. The terms “NSCLC” and “squamous cell carcinoma of the lung” are well known in the art and the skilled person knows how to determine the type of NSCLC (e.g., whether the NSCLC has a squamous or non-squamous histology), e.g., by biopsy (e.g., transbronchial biopsy (TBB), computed tomography -guided needle biopsy (CTNB)), imaging methods (e.g., computer tomography (CT), positron emission tomography (PET), positron emission tomography-computed tomography (PET-CT), X-ray, magnetic resonance imaging (MRI)), ultrasound methods, bronchoscopy (e.g., endobronchial ultrasound (EBUS) bronchoscopy), cytology (e.g., sputum cytology), thoracentesis, and thoracoscopy. The main methods to diagnose whether the NSCLC is squamous or non-squamous are histology and / or cytology of tumor samples obtained via broad diagnostic methods (biopsy (e.g., TBB and CTNB), bronchoscopy (e.g., EBUS bronchoscopy), sputum cytology, thoracentesis, and thoracoscopy). Imaging methods (such as PET, CT, PET-CET, X ray, and MRI) may only be used to diagnose lung cancer (e.g., NSCLC) but not a histologic subtype.

[0088] The term “CTLA4” or “CTLA-4” relates to cytotoxic T lymphocyte antigen-4. “CTLA4” or “CTLA-4” are used interchangeably herein. CTLA4 is expressed on the surface of cells and, if it is located at the surface of said cells, is accessible to binding by CTLA4 specific antibodies. “Cell surface” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. For example, a transmembrane protein having one or more extracellular portions is considered as being expressed on the cell surface.

[0089] The term “immune cell” means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., a bacterial or viral infection or an auto-antigen). In some embodiments, an immune cell is a leukocyte, such as a white blood cell. Immune cells

[0090] [P2025W001] / / 241356WO include neutrophils, eosinophils, basophils, lymphocytes, and / or monocytes. Lymphocytes include T lymphocytes (T cells) and B lymphocytes (B cells). Immune cells can also be dendritic cells, natural killer (NK) cells, and / or a mast cell.

[0091] The term “antibody” refers to a molecule that possesses an antigen-binding site. The antibody can comprise a “variable region”. The terms “variable region” and “variable domain” are used interchangeably herein. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region comprises a “hypervariable region” whose residues are responsible for antigen binding, and as used herein means the segment of an antibody which contains three CDRs, designated CDR1, CDR2 and CDR3. A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable region of the heavy chain may be referred to as “ VH.” The variable region of the light chain may be referred to as “VL.” Typically, the variable regions of both the heavy and light chains comprise three hypervariable regions, the CDRs, which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain. The variable domains of the heavy and light chains each contain three CDRs, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; Kabat et al., J Biol Chem 1977,

[0092] [P2025W001] / / 241356WO 252: 6609-6616; Kabat, Adv Prot Chem 1978, 32: 1-75), the Chothia numbering system (Chothia & Lesk, J Mol Biol 1987, 196: 901-917; Chothia et al., Nature 1989, 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev Comparat Immunol 2003, 27: 55-77). For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (Lefranc et al., Dev Comparat Immunol 2003, 27: 55-77). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region or CRR residues as herein defined. “Antibody” includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single chain antibodies, disulfide- linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies disclosed herein). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG2, IgGs, IgG4, IgAi and IgA2) or subclass.

[0093] The term “antigen-binding fragment” or “antibody -binding portion” of an antibody refers to one or more portions or fragments of an antibody that contain the antibody’s CDRs and optionally the framework residues that comprise the antibody’s variable domain antigen recognition site, and exhibit an ability to immunospecifically bind an antigen. Examples of antibody fragments encompassed within the term “antigen binding fragment” or “antigen binding portion” include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab’)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., 1989, Nature 341 :544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to

[0094] [P2Q25WO01] / / 241356WO form 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). Such single chain antibodies are also intended to be encompassed within the term “antigenbinding fragment” or “antibody-binding portion” of an antibody. Also included within the term “antigen-binding” fragment of an antibody are a rlgG fragments consisting of one heavy chain (HC) and one light chain (LC), i.e., consisting of one of the identical halves of an IgG antibody. A further example are binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding-domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication Nos. 2003 / 0118592 and 2003 / 0133939.

[0095] These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. “Antigen-binding fragment” or “antibody-binding portion” may further include fusion proteins comprising the antibody’s variable region antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). As used within the present invention, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0096] [P2025W001] / / 241356WO The term “human antibody” refers to an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” refer to an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

[0097] A “humanized antibody” is an immunoglobulin comprising a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The nonhuman immunoglobulin providing the CDR’s is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”. Constant regions need not be present, but if they are, they preferably can be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody can be an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human. One says that the donor antibody has been “humanized”, by the process of “humanization”, because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR’s. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs

[0098] [P2025W001] / / 241356WO are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an Fc.gamma.RIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations).

[0099] An antibody that “specifically binds to” or “immunospecifically binds to” a specified target protein is an antibody that exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g., without producing undesired results such as false positives. Antibodies, or binding fragments thereof, useful in the present invention will bind to the target protein with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins. As used herein, an antibody is said to bind specifically to a polypeptide comprising a given amino acid sequence, e.g., the amino acid sequence of a mature human CTLA4 molecule, if it binds to polypeptides comprising that sequence but preferably does not bind to proteins lacking that sequence.

[0100] “Chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0101] The term “epitope” refers to the part of an antigen that as used herein, refers to an agent that elicits an immune response; and / or an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes

[0102] [P2025W001] / / 241356WO usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0103] The term “antigen” as used herein, refers to an agent that elicits an immune response; and / or an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer]) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0104] The terms “chemotherapeutic agent” or “chemotherapeutical agent” or “chemotherapy agent” can be used interchangeably herein. A chemotherapeutic agent is a chemical compound useful in the treatment of cancer. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antitumour antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs), anti-progesterones, estrogen receptor

[0105] [P2025W001] / / 241356WO down-regulators (ERDs), estrogen receptor antagonists, leutinizing hormone-releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, anti-sense oligonucleotides that that inhibit expression of genes implicated in abnormal cell proliferation or tumor growth. Chemotherapeutic agents include cytostatic and / or cytotoxic agents. Chemotherapeutic agents as used herein do not include antibodies.

[0106] The term “chemotherapy treatment” as used herein refers to the treatment of a disease, e.g., a cancer such as NSCLC, in particular NSCLC with squamous histology, with a chemotherapy agent.

[0107] The term “administration route” refers to the means of administration of an active agent or a pharmaceutical composition. The terms “active agent” or “therapeutically effective agent” as used herein refer to compounds that exert a pharmaceutical effect in the treatment of a disease. The anti-CTLA4 antibody or fragment thereof as disclosed herein can be administered by any administration route known to the skilled person. In general, active agents or pharmaceutical compositions can be administered using any suitable enteral route or parenteral route of administration. The term “enteral route” of administration refers to the administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal, and rectal route, or intragastric route. “Parenteral route” of administration refers to a route of administration other than enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumour, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal, subcutaneous, or topical administration. For example, anti-CTLA4 antibody or fragment thereof of the present disclosure can be administered using different enteral or parenteral administration routes. The anti-CTLA4 antibody or fragment thereof of the present disclosure can also be administered using the same administration route, e.g., intravenous administration using an intravenous injection or intravenous infusion.

[0108] The term “separate administration” as used herein refers to the administration of at least two active agents or therapeutically effective agents using different pharmaceutical compositions

[0109] [P2025W001] / / 241356WO or formulations. For example, a first active agent and a second active agent can be administered using two different pharmaceutical compositions, each comprising either the first active agent or the second active agent.

[0110] The terms “dosage” or “dose” are used interchangeably herein and refer to the amount of an active agent or therapeutically effective agent that is administered to a subject in a single administration step. For example, the anti-CTLA4 antibody or fragment thereof as described herein can be administered in a dosage of about 1 mg / kg body weight, about 3 mg / kg body weight, about 6 mg / kg body weight, about 10 mg / kg body weight, about 12 mg / kg body weight, about 15 mg / kg body weight, or about 20 mg / kg body weight.

[0111] The term “treatment cycle” as used herein refers to the repetitive treatment of a subject with an active agent, therapeutically effective agent, or pharmaceutical composition, or combinations thereof. A treatment cycle begins with the administration of a first dosage or first concurrent administration of more than one dosage of an active agent, therapeutically effective agent, or pharmaceutical composition, or combinations thereof and ends either with the administrations of a consecutive or subsequent dosage of the same or another active agent, therapeutically effective agent, or pharmaceutical composition, or combinations thereof or with the end of the treatment. In some embodiments a treatment cycle has at least about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days. In some embodiments, a treatment cycle has 21 days.

[0112] The terms “platinum-containing chemotherapy” or “platinum-based chemotherapy” refer to the use of chemotherapeutic agent(s) (also known as platins) for treating cancer that are coordination complexes of platinum. Platinum-containing chemotherapeutic agents are alkylating agents that crosslink DNA, resulting in ineffective DNA mismatch repair and generally leading to apoptosis. Examples of platins include cisplatin, carboplatin, and oxaliplatin.

[0113] [P2025W001] / / 241356WO The term “monotherapy” as used herein refers to the treatment of a particular disease or condition with a single active agent or single therapeutically effective agent. For example, the anti-CTLA4 antibody or fragment thereof disclosed herein can be administered as a monotherapy for the treatment of NSCLC, particularly for the treatment of NSCLC with a squamous histology, meaning that no additional active agents directed to the treatment of NSCLC are administered during the treatment method.

[0114] The term “immunotherapy” as used herein refers to the modification of the subject's immune system for the treatment of a disease or condition. Immunotherapy can comprise the activation, stimulation, or suppression of the subject's immune system. “Immunotherapeutic agents” are active agents that are capable of modifying the subject's immune system, e.g., by activating, stimulating, or suppressing the subject's immune system.

[0115] The term “overall survival” (OS) as used herein refers to the time period between the randomization and the death of the subject by any cause.

[0116] The term “progression-free survival” (PFS) as used herein refers to the time period between the randomization and the first objective tumor progression according to RECIST vl.1 or death from any cause, whichever occurs first.

[0117] The terms “effective amount” or “therapeutically effective amount” refer to an amount of a given substance that is sufficient in quantity to produce a desired effect, including an improvement or remediation of the disease, disorder, or symptoms of the disease or condition.

[0118] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended

[0119] [P2025W001] / / 241356WO merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0120] The anti-CTLA4 antibody for use in treating non-small cell lung cancer

[0121] The present invention provides an anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology. In some embodiments, an anti-CTLA4 antibody is provided for use in a method of treating NSCLC in a subject in need thereof, wherein the NSCLC has a squamous histology.

[0122] The present invention provides antibodies or fragments thereof that immunospecifically bind to CTLA4, in particular human CTLA4, preferably expressed on the surface of a cell at an endogenous or transfected concentration. Cytotoxic T lymphocyte antigen-4 (CTLA4) is a regulator of adaptive immune responses, having a role in the maintenance of peripheral tolerance and in shaping the repertoire of emergent T cell responses. CTLA4 is expressed on the surface of cells and, if it is located at the surface of said cells, is accessible to binding by CTLA4-specific antibodies.

[0123] In some embodiments, the anti-CTLA4 antibody or fragment thereof is capable of binding to human CTLA4. In some embodiments, CTLA4 is expressed on the surface of a T cell. In some embodiments, the anti-CTLA4 antibody or the anti-CTLA4 antibody fragment is a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody, or a fragment thereof. In some embodiments, the anti CTLA4 antibody or fragment thereof is a humanized anti-CTLA4 antibody or a fragment thereof. In some embodiments, the anti CTLA4 antibody or fragment thereof is a humanized anti-CTLA4 IgGl monoclonal antibody or a fragment of a humanized anti-CTLA4 IgGl monoclonal antibody.

[0124] In some embodiments, the anti-CTLA4 antibody fragment is an antigen-binding fragment or a variant thereof. In some embodiments, the antigen binding fragment or variant thereof is

[0125] [P2025W001] / / 241356WO selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a rlgG, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof.

[0126] Without wishing to be bound by theory, CTLA4 is contemplated to be recycled between the cell surface and endosomes, where it is usually prevented from lysosomal degradation and can recycle back to the cell surface by binding to the lipopolysaccharide-responsive and beige-like anchor (LRBA) protein. The anti-CTLA4 antibodies and fragments thereof disclosed herein have reduced autoimmune side effects when used to enhance immune responses and are particularly suitable for treating cancer in subjects, particularly lung cancer, preferably NSCLC, more preferably NSCLC with squamous histology.

[0127] In some embodiments, the subject in need for cancer treatment is a human.

[0128] In some embodiments, the anti-CTLA4 antibody or fragment thereof is the only immunotherapeutic agent administered to the subject. This means that no additional immunotherapeutic agents are administered to the subject in parallel to the anti-CTLA4 antibody.

[0129] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered as a monotherapy.

[0130] Surprisingly, the anti-CTLA4 antibody shows an unexpected beneficial effect in the treatment of NSCLC with squamous histology, while in parallel providing a safe treatment. In particular, the anti-CTLA4 antibody shows good safety profiles in subjects afflicted with NSCLC with squamous histology, particularly in subjects with primary or acquired resistance to immune checkpoint inhibitor cancer immunotherapy and / or chemotherapy and without targetable NSCLC driver mutations (except KRAS mutations). Furthermore, immune-related adverse events are reduced by the anti-CTLA4 antibody therapy of the invention. Immune- related adverse events such as skin rash, hepatitis, colitis and endocrinopathies, particularly hypopituitarism are characteristic side effects of anti-cancer therapies based on immune

[0131] [P2025W001] / / 241356WO checkpoint inhibitors such as anti-CTLA4 antibodies. It was therefore surprising that the anti- CTLA4 antibody therapy of the invention improves the efficiency of the anti-cancer treatment while in parallel reducing undesired side effects, e.g., immune-related adverse events.

[0132] In some embodiments, the NSCLC has a squamous histology (squamous NSCLC) and has a defect in PTEN tumour suppressor function. This defect can be at the transcriptional, posttranscriptional, translational, or posttranslational level or a combination thereof. A PTEN (phosphatase and tensin homologue) defect in squamous NSCLC is contemplated to lead to the enrichment of regulatory T cells (Treg). High Treg enrichment can be a poor prognostic marker for the treatment of squamous NSCLC. The anti-CTLA4 antibody is thus suggested to lead to an effective tumor-infiltrating Treg-depletion and therefore efficient anti -cancer treatment in squamous NSCLC, in particular in squamous NSCLC with defects in PTEN.

[0133] The anti-CTLA4 antibody

[0134] In some embodiments, the anti-CTLA4 antibody or fragment thereof is capable of binding to human CTLA4. In some embodiments, the invention provides an anti-CTLA4 antibody, which is capable of binding to human CTLA4, for use in a method of treating NSCLC in a subject in need thereof, wherein the NSCLC has a squamous histology. The term “binding” preferably relates to a specific binding. An antibody usually is capable of binding to a predetermined target if it has a significant affinity for said predetermined target and binds to said predetermined target in standard assays. “Affinity” or “binding affinity” is often measured by equilibrium dissociation constant (KD). Preferably, the term “significant affinity” refers to the binding to a predetermined target with a dissociation constant (KD) of 10'5M or lower, 10'6M or lower, 10'7M or lower, 10'8M or lower, or 10'9M or lower. An antibody is not (substantially) capable of binding to a target if it has no significant affinity for said target and does not bind significantly, in particular does not bind detectably, to said target in standard assays. For example, if the KD for binding of an antibody to the target to which the antibody is capable of binding is 10'9M, the KD for binding to a target for which the antibody

[0135] [P2025W001] / / 241356WO has no significant affinity would be is at least around 10'8M, 10'7M, 10'6M, IO'5M, 10'4M, IO’3M, 10’2M, or IO'1M.

[0136] Binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., Antibody- Antigen Interactions In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein. Affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data may be analyzed, for example, by the method of Scatchard et al. (Scatchard et al., Ann NY Acad ScL 1949, 51 :660). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g., KD, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer. For example, the affinity of an antibody can be evaluated by Octet. Multi-concentration kinetic experiments can be performed on the Octet Red96 system (ForteBio). Anti-hlgG Fc biosensors (ForteBio, #18-5064) can be hydrated in sample diluent (0.1% BSA in PBS and 0.02% Tween 20) and preconditioned in pH 1.7 glycine. The antigen can be diluted using a 7-point, 2-fold serial dilution starting at 600 nM with sample diluent. The antibody to be tested can be diluted to 10 pg / mL with sample diluent and then immobilized onto anti-hlgG Fc biosensors for 120 seconds. After baselines are established for 60 seconds in sample diluent, the biosensors can be moved to wells containing the antigen at a series of concentrations to measure the association. Association can be observed (e.g., for 120 seconds) and dissociation can be observed (e.g., for 180 seconds) for each protein of interest in the sample diluent. The binding affinities can be characterized by fitting the kinetic sensorgrams to a monovalent binding model (1 : 1 binding).

[0137] The anti-CTLA4 antibody or fragment thereof disclosed herein is specific for CTLA4 if it is capable of binding to CTLA4 but is not (substantially) capable of binding to other targets.

[0138] [P2025W001] / / 241356WO The anti-CTLA4 antibody or fragment thereof disclosed herein preferably does not inhibit binding of human CTLA4 to the B7.1 (CD80) and B7.2 (CD86) ligands of antigen presenting cells. In some embodiments, the level of B7.1 and B7.2 on immune cells following anti- CTLA4 treatment is used as a biomarker for measuring the biological activity of anti-CTLA4 antibodies in vivo and monitoring responses to anti-CTLA4 treatment by measuring the level B7.1 and / or B7.2 expression on immune cells, and comparing the level of expression before and after treatment. In some embodiments, the level of B7.1 and / or B7.2 expression is monitored over time during a course of therapy. The therapeutic effect of CTLA4 antibodies disclosed herein is preferably achieved through antibody -mediated depletion of Tregs specifically within tumor microenvironment. The anti-CTLA4 antibodies disclosed herein are preferably not capable of blocking B7-CTLA4 interactions under physiological conditions.

[0139] A fundamental question for the generation of safe and effective anti-CTLA4 antibodies is whether cancer immunotherapeutic effects (CITE) and immunotherapy -related adverse effects (irAE) are intrinsically linked. The classical checkpoint blockade hypothesis stipulated that anti-CTLA4 antibodies promote cancer immunity by blocking a negative signal of B7- CTLA4 interactions to promote naive T cell activation in the lymphoid organ. According to this model, therapeutic antibodies are antagonists that functionally inactivate CTLA4-B7 interactions. Since genetic inactivation of CTLA4 expression leads to autoimmune diseases in mouse and human, it was assumed that the irAE would be a necessary price for CITE. However, there is no evidence to date that blocking CTLA-4 interaction with B7.1 and B7.2 is either necessary or sufficient for the CITE of anti-CTLA-4 antibodies. Without wishing to be bound by theory, it is contemplated that selective depletion of Tregs in the tumor microenvironment constitutes the main mechanism of action of anti-CTLA4 antibodies. It is not relevant whether an antibody is capable of blocking B7-CTLA4 interactions under physiological conditions for the induction of CITE. In some embodiments, the anti-CTLA4 antibody or fragment thereof disclosed herein can induce CITE without blocking B7-CTLA4 interactions.

[0140] [P2025W001] / / 241356WO In some embodiments, the anti-CTLA4 antibody is not ipilimumab (marketed as YERVOY®).

[0141] In some embodiments, the anti-CTLA4 antibody is described in International Patent Application Publication No., WO 2017 / 106372, which is incorporated herein in its entirety.

[0142] In some embodiments, the anti-CTLA antibody fragment is an antigen-binding fragment or a variant thereof. In some embodiments, the antigen binding fragment or variant thereof is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a rlgG, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof.

[0143] In some embodiments, the anti-CTLA4 antibody or fragment thereof comprises: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1, (2) a complementarity-determining region 2 (HCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2, and (3) a complementarity-determining region 3 (HCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain variable region comprising (1) a complementarity-determining region 1 (LCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4, (2) a complementarity-determining region 2 (LCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 5, and (3) a complementarity-determining region 3 (LCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 6.

[0144] In some embodiments, the anti-CTLA4 antibody or fragment thereof comprises:

[0145] [P2025W001] / / 241356WO a) a heavy chain variable domain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7; and b) a light chain variable domain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8.

[0146] In some embodiments, the antibody or fragment thereof having the ability to bind to CTLA4 can be a polyclonal, monoclonal antibody or a chimeric antibody, or fragment thereof, optionally having an IgG or IgM isotype of any subclass, such as subclass I. Preferably, the anti-CTLA4 antibody is an IgG or IgM antibody, preferably an IgG antibody, more preferably an IgGl, IgG2, IgG3 or IgG4 antibody, even more preferably and IgGl antibody. The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity. In some embodiments, the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e.g., mouse, fused to an immortalized cell.

[0147] In some embodiments, the anti-CTLA4 antibody or fragment thereof comprises a constant region. In some embodiments, the constant region of the anti-CTLA4 antibody is derived from a human antibody. In some embodiments, the constant region or fragment of the constant region of the anti-CTLA4 antibody fragment is derived from a human antibody. In some embodiments, the constant region of the anti-CTLA4 antibody is selected from the constant region of human IgGl, IgG2, IgG3 or IgG4. In some embodiments, the constant region of the anti-CTLA4 antibody is human a IgGl constant region. In some embodiments, the constant region of the anti-CTLA4 antibody is a human IgG2 constant region. In some embodiments, the constant region of the anti-CTLA4 antibody is a human IgG3 constant region. In some embodiments, the constant region of the anti-CTLA4 antibody is a human IgG4 constant region. In some embodiments, the constant region or fragment of the constant

[0148] [P2025W001] / / 241356WO region of the anti-CTLA4 antibody fragment is selected from the constant region or a fragment thereof of human IgGl, IgG2, IgG3 or IgG4. In some embodiments, the constant region or fragment of the constant region of the anti-CTLA4 antibody fragment is a human IgGl constant region or fragment thereof. In some embodiments, the constant region or fragment of the constant region of the anti-CTLA4 antibody fragment is a human IgG2 constant region or fragment thereof. In some embodiments, the constant region or fragment of the constant region of the anti-CTLA4 antibody fragment is a human IgG3 constant region or fragment thereof. In some embodiments, the constant region or fragment of the constant region of the anti-CTLA4 antibody fragment is a human IgG4 constant region or fragment thereof.

[0149] In some embodiments, the anti-CTLA4 antibody or fragment thereof comprises an IgGl Fc region (or Fc domain). In some embodiments, the anti-CTLA4 antibody or fragment thereof comprises an IgGl Fc comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9. In some embodiments, the anti-CTLA4 antibody can comprise a mutated Fc region. Relative to the sequence of the IgGl backbone in SEQ ID NO: 14, the mutation may be M135Y, S137T, T139E, S181A, E216A, or K217A, or a combination thereof. These mutations are contemplated to lead to increased antibody dependent cellular cytotoxicity (ADCC) and increased half-life of the antibody in vivo.

[0150] In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

[0151] [P2025W001] / / 241356WO In some embodiments, the anti-CTLA4 antibody comprises: a) a heavy chain whose amino acid sequence is encoded by a nucleotide sequence set forth in SEQ ID NO: 11, or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11; and b) a light chain whose amino acid sequence is encoded by a nucleotide sequence set forth in SEQ ID NO: 13, or a nucleotide sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.

[0152] In some embodiments, the thymidine in the nucleotide sequences set forth in SEQ ID NO: 11 and 13 is uridine. Meaning, all thymidines present in the nucleotide sequences of SEQ ID NO: 11 and 13 can be uridines.

[0153] In some embodiments, the anti-CTLA4 antibody is encoded by one or more nucleic acid molecules.

[0154] In some embodiments, the anti-CTLA4 antibody or fragment thereof is a pH-sensitive anti- CTLA4 antibody or a pH-sensitive anti-CTLA4 antibody fragment. For example, the pH- sensitive anti-CTLA4 antibody or anti-CTLA4 antibody fragment can dissociate from CTLA4 at pH 6.5 or below, more preferably pH 5.5 or below. In some embodiments, binding to CTLA4 is reduced at an endosomal pH of 5.5 by more than 50% relative to binding at neutral pH (pH 7.0). Such a reduction may reach more than 75% at lysosomal pH 4.5 as compared to pH 7.0. The antibody- antigen complex preformed at pH 7.0 may dissociate under an acidic environment of pH 4.5-6.0. The reduction in binding may also be in comparison to a reference antibody which may be considerably less pH sensitive using the same standard. The reference antibody may be an antibody known in the art such as Ipilimumab or Tremelimumab. In the context of an engineered antibody or antibody fragment, the changes may also be in comparison to a wild-type antibody or antibody fragment which may be considerably less pH sensitive using the same standard. Without wishing to be bound by theory, it is contemplated

[0155] [P2025W001] / / 241356WO that a pH-sensitive antibody or antibody fragment is not only safer but also more effective in Treg depletion and tumor rejection than a pH-insensitive CTLA4 antibody (e.g., Ipilimumab) or a fragment thereof. Meaning, Ipilimumab can bind to CTLA4 at a pH of 4-7 and no dissociation can be observed at pH 4-7. pH-insensitive antibodies can cause down-regulation of CTLA4 through lysosomal degradation. CTLA4 down-regulation can cause autoimmune diseases, while in the tumor CTLA4 down-regulation can reduce ADCC activity and thus anti-cancer efficacy. The sensitivity to pH can be measured by any method known to the skilled person. For example, human or monkey-CTLA4-Fc or (0.5 pg / ml) can be coated on ELISA plates at 4°C overnight. Biotinylated anti-CTLA4 antibodies anti-CTLA4 antibody fragments can be added at 1 pg / ml in 1% BSA PBS with pH 4.5-7.0. Two hours later, antibodies and antibody fragments binding with or to CTLA4 can be measured by using HRP- labeled streptavidin. A pH-sensitive anti-CTLA4 antibody or fragment thereof can comprise, for example, the following CDR sequences: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1, (2) a complementarity-determining region 2 (HCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2, and (3) a complementarity-determining region 3 (HCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain variable region comprising (1) a complementarity-determining region 1 (LCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4, (2) a complementarity-determining region 2 (LCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 5, and (3) a complementarity-determining region 3 (LCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 6.

[0156] Although the following provides considerations regarding the mechanism underlying the therapeutic efficacy of antibodies of the disclosure it is not to be considered as limiting to the

[0157] [P2025W001] / / 241356WO invention in any way. In some embodiments, the antibodies described herein are contemplated to be a highly selective, humanized monoclonal immunoglobulin G1 (IgGl)-kappa isotype antibody against CTLA4 with a robust anti-tumor activity and lower autoimmune toxicity in comparison to ipilimumab. The disclosed antibodies preferably can dissociate from CTLA4 under low pH in endosomes to allow both CTLA4 and the antibody to escape from lysosomal degradation and recycle to the cell surface. Unlike ipilimumab that down-regulates CTLA4 expression on Treg cells, the antibodies can keep a high-level expression of CTLA4 on Treg cells through this recycling mechanism and makes Treg cells a better target for antibodydependent cellular cytotoxicity, particularly in the tumor microenvironment (TME). The selective elimination of Treg cells in the tumor microenvironment and maintenance of CTLA4 expression in Treg cells in the peripheral tissues by the anti-CTLA4 antibody is contemplated to form the cellular and molecular basis for more potent tumor rejection and low toxicity. For example, the anti-CTLA4 antibody described herein dissociates from CTLA4 in endosomes, allows normal recycling of both antibodies and CTLA4, which lead to a much-reduced autoimmune toxicity. The preservation of the recycling of both CTLA4 and the anti-CTLA4 antibody facilitates more potent ADCC to eliminate Treg cells in the tumor microenvironment and induces strong CITE. ADCC preferably occurs when antibodies bind to antigens such as CTLA4 on Treg cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells.

[0158] In some embodiments, the anti-CTLA4 antibody comprises one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein and comprises said CDRs together with their intervening framework regions. Construction of antibodies made by recombinant DNA techniques may result in the introduction of residues N- or C-terminal to the variable regions encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable regions to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels. In one embodiment an antibody comprising one or more CDRs, a set of CDRs or a combination of sets of CDRs as described herein comprises said CDRs in a human antibody framework.

[0159] [P2025W001] / / 241356WO It will be appreciated by those skilled in the art that in particular the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to bind CTLA4. For example, CDR regions will be either identical or highly homologous to the regions of antibodies specified herein. By “highly homologous” it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be modified so that they show substantial homology with the regions of antibodies specifically disclosed herein.

[0160] It will be appreciated by those skilled in the art that the anti-CTLA4 antibody can comprise variants of the sequence(s) disclosed herein without losing the ability to bind CTLA4. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

[0161] Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place.

[0162] Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine,

[0163] [P2025W001] / / 241356WO tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0164] Antibodies and antibody fragments described herein and useful in the methods described herein can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 1975, 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes.

[0165] In some embodiments, an animal system for preparing hybridomas that secrete monoclonal antibodies may be a murine system. Hybridoma production in the mouse is a very well- established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0166] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (e.g., described in Spieker-Polet et al., Proc Natl Acad Sci U.S.A. 1995, 92: 9348; see also Rossi et al., Am J Clin Pathol 2005, 124: 295).

[0167] Yet another strategy for generating monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes producing antibodies of defined specificity. For details of recombinant antibody engineering see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Bemiy K.C. Lo Antibody Engineering ISBN 1 - 58829- 092-1.

[0168] To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from the antigen sequence, i.e., the sequence against which the antibodies are to be directed, an enriched preparation of recombinantly expressed antigen or fragments thereof and / or cells

[0169] [P2025W001] / / 241356WO expressing the antigen, as described. Alternatively, mice can be immunized with nucleic acid encoding the antigen or fragments thereof. In the event that immunizations using a purified or enriched preparation of the antigen do not result in antibodies, mice can also be immunized with cells expressing the antigen, e.g., a cell line, to promote immune responses.

[0170] The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusions. Mice can be boosted intraperitonealy or intravenously with antigen expressing cells 3 days before sacrifice and removal of the spleen to increase the rate of specific antibody secreting hybridomas.

[0171] To generate hybridomas producing monoclonal antibodies, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody secreting hybridomas. By Immunofluorescence and FACS analysis using antigen expressing cells, antibodies with specificity for the antigen can be identified. The antibody secreting hybridomas can be re-plated, screened again, and if still positive for monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization. Antibodies also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as are well known in the art (Morrison, Science 1985, 229: 1202).

[0172] For example, in some embodiments, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in International Patent Application Publication Nos. WO 87 / 04462 and WO 89 / 01036 and EP 338 841 A or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO cells, NS / 0 cells, HEK293T cells or HEK293 cells or

[0173] [P2025W001] / / 241356WO alternatively other eukaryotic cells like plant derived cells, fungal or yeast cells. The method used to introduce these genes can be methods described in the art such as electroporation, lipofectine, lipofectamine or others. After introduction of these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0174] Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g., E. coli. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see e.g., Verma, R., et al., J Immunol Meth 1998,216: 165-181; Pollock, et al., J Immunol Meth 1999, 231 : 147-157; and Fischer, R., et al., Biol Chem 1999, 380: 825-839.

[0175] Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al., Nature 1998, 332:323-327; Jones et al., Nature 1986, 321 :522-525; and Queen et al., Proc Natl Acad Sci USA 1989, 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region.

[0176] [P2025W001] / / 241356WO The ability of antibodies to bind an antigen can be determined using standard binding assays (e.g., ELISA, Western Blot, Immunofluorescence and flow cytometric analysis).

[0177] To purify antibodies, selected hybridomas can be grown in two-liter spinner- flasks for monoclonal antibody purification. Alternatively, antibodies can be produced in dialysis-based bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography with protein G-sepharose or protein A-sepharose. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0178] To determine if selected monoclonal antibodies bind to unique epitopes and / or to characterize one or more binding properties, site-directed or multi-site directed mutagenesis can be used.

[0179] To determine the isotype of antibodies, isotype ELISAs with various commercial kits (e.g., Zymed, Roche Diagnostics) can be performed. Wells of microtiter plates can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either mouse IgGl, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg / ml) and analyzed at OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) may be used as described by the manufacturer.

[0180] In order to demonstrate presence of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing antigen, flow cytometry can be used. Cell lines expressing naturally or after transfection antigen and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1 % FBS, and can be incubated at 4 °C for 30 min. After washing, the APC- or Alexa647-labeled

[0181] [P2025W001] / / 241356WO anti IgG antibody can bind to antigen-bound monoclonal antibody under the same conditions as the primary antibody staining. The samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate on single, living cells. In order to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, the method of co-transfection can be employed. Cells transiently transfected with plasmids encoding antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the majority of transfected cells express both transgenes, antigen- specific monoclonal antibodies bind preferentially to fluorescence marker expressing cells, whereas non-specific antibodies bind in a comparable ratio to non-transfected cells. An alternative assay using fluorescence microscopy may be used in addition to or instead of the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy.

[0182] In order to demonstrate presence of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing antigen, immunofluorescence microscopy analysis can be used. For example, cell lines expressing either spontaneously or after transfection antigen and negative controls lacking antigen expression are grown in chamber slides under standard growth conditions in DMEM / F12 medium, supplemented with 10 % fetal calf serum (FCS), 2 mM L-glutamine, 100 lU / ml penicillin and 100 pg / ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against the antigen for 30 min. at 25°C. After washing, cells can be reacted with an Alexa555-labelled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

[0183] Cell extracts from cells expressing antigen and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes,

[0184] [P2025W001] / / 241356WO blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

[0185] Antibodies can be further tested for reactivity with antigen by Immunohistochemistry in a manner well known to the skilled person, e.g., using paraformaldehyde or acetone fixed cryosections or paraffin embedded tissue sections fixed with paraformaldehyde from noncancer tissue or cancer tissue samples obtained from patients during routine surgical procedures or from mice carrying xenografted tumors inoculated with cell lines expressing spontaneously or after transfection antigen. For immunostaining, antibodies reactive to antigen can be incubated followed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the vendors instructions. The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models.

[0186] The treatment

[0187] In some embodiments, the anti-CTLA4 antibody or fragment thereof is the only immunotherapeutic agent administered to the subject. This means that no additional immunotherapeutic agents are administered to the subject in parallel to the anti-CTLA4 antibody.

[0188] Ins some embodiments, the anti-CTLA4 antibody is administered as a monotherapy. For example, when the anti-CTLA4 antibody or fragment thereof is administered as a monotherapy for the treatment of NSCLC, particularly for the treatment of NSCLC with a squamous histology, no additional active agents directed to the treatment of NSCLC are administered in parallel.

[0189] In some embodiments, the subject in need thereof is a human.

[0190] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered at a dosage ranging from about 0.1 mg / kg to about 45 mg / kg body weight. In some embodiments,

[0191] [P2025W001] / / 241356WO the anti-CTLA4 antibody or fragment thereof is administered at a dosage ranging from about 1 mg / kg to about 30 mg / kg body weight. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered at a dosage ranging from about 2 mg / kg to about 20 mg / kg body weight.

[0192] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered at a dosage independently selected from about 1 mg / kg body weight, about 3 mg / kg body weight, about 6 mg / kg body weight, about 10 mg / kg body weight, about 12 mg / kg body weight, about 15 mg / kg body weight, and about 20 mg / kg body weight.

[0193] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered at a dosage independently selected from about 3 mg / kg body weight, about 6 mg / kg body weight, and about 10 mg / kg body weight.

[0194] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered at a dosage of 3 mg / kg body weight. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered at a dosage of 6 mg / kg body weight. In some embodiments, the anti- CTLA4 antibody or fragment thereof is administered at a dosage of 10 mg / kg body weight.

[0195] The anti-CTLA4 antibody or fragment thereof as described herein is administered in treatment cycles. In some embodiments, a treatment cycle has at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, or about 28 days.

[0196] In some embodiments, a treatment cycle has treatment cycle has about 18 to about 28 days. In some embodiments, a treatment cycle has about 18 to about 24 days. In some embodiments, a

[0197] [P2025W001] / / 241356WO treatment cycle has about 18 to about 21 days. In some embodiments, a treatment cycle has about 21 days. In some embodiments, a treatment cycle has 21 days.

[0198] In some embodiments, a treatment cycle is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In some embodiments, a treatment cycle is repeated 1,

[0199] 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In some embodiments a treatment cycle is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 times. However, the treatment can be continued until the subject is free or essentially free of cancer or a new anti-cancer treatment is started. The treatment can also be continued once the first objective tumor progression according to RECIST vl.1 is determined in the subject.

[0200] In some embodiments, a treatment cycle is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 times. In some embodiments, a treatment cycle is repeated at least 17 times. In some embodiments, a treatment cycle is repeated 17 times.

[0201] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered about every 6 weeks. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered every 6 weeks. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered about every 4 weeks. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered every 4 weeks. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered about every 3 weeks. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered every 3 weeks.

[0202] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered for 1, 2,

[0203] 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 treatment cycles. In some embodiments, anti-CTLA4 antibody or fragment thereof is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 treatment cycles. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered for 17 treatment cycles or up to 1 year. In some embodiments, the anti-CTLA4

[0204] [P2025W001] / / 241356WO antibody or fragment thereof is administered for 17 treatment cycles. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered for up to 1 year.

[0205] In some embodiments, the subject is treated with one or more dosages of the anti-CTLA4 antibody or fragment thereof per treatment cycle. In some embodiments, the subject is treated with 1, 2, 3, 4, 5 ,6 ,7, 8, 9, or 10 dosages of the anti-CTLA4 antibody or fragment thereof per treatment cycle. In some embodiments, the subject is treated with 1 dosage of the anti-CTLA4 antibody or fragment thereof per treatment cycle. In some embodiments, the subject is treated for 17 treatment cycles with 1 dosage of the anti-CTLA4 antibody per treatment cycle.

[0206] In some embodiments, the subject is treated with a fixed dosage of the anti-CTLA4 antibody or fragment thereof throughout the treatment. In some embodiments, the subject is treated with a fixed dosage of the anti-CTLA4 antibody or fragment thereof in the individual treatment cycles. In such embodiments, the dosage of the anti-CTLA4 antibody or fragment thereof does not change between the individual treatment cycles. In some embodiments, the subject is treated with 1 dosage of the anti-CTLA4 antibody or fragment thereof per treatment cycle and the dosage does not change between the individual treatment cycles.

[0207] In some embodiments, the subject is treated with 1 dosage of about 3, about 6, or about 10 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated with 1 dosage of 3, 6, or 10 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated with 1 dosage of about 6 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated with 1 dosage of 6 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated with 1 dosage of about 10 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some

[0208] [P2025W001] / / 241356WO embodiments, the subject is treated with 1 dosage of 10 mg / kg body weight of the anti- CTLA4 antibody per treatment cycle.

[0209] In some embodiments, the subject is treated for up to 17 consecutive treatment cycles or up to 1 year with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 17 consecutive treatment cycles with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for 17 consecutive treatment cycles with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 1 year with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle.

[0210] In some embodiments, the subject is treated for up to 17 consecutive treatment cycles or up to 1 year with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 1 year with 1 dosage of 3 mg / kg body weight of the anti- CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 17 consecutive treatment cycles or up to 1 year with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for up to 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle. In some embodiments, the subject is treated for 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle.

[0211] [P2025W001] / / 241356WO In some embodiments, the subject is treated with at least 1 dosage of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof. In some embodiments, the subject is treated with at least 2 dosages of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof. In some embodiments, the subject is treated with 2 dosages of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof.

[0212] In some embodiments, the subject is treated with different dosages of the anti-CTLA4 antibody or fragment thereof throughout the treatment. In some embodiments, the subject is treated with different dosages of the anti-CTLA4 antibody or fragment in the individual treatment cycles. In such embodiments, the dosage changes between the individual treatment cycles. For example, the treatment can be initiated with a high dosage of the anti-CTLA4 antibody or fragment thereof and can be continued with a lower dosage compared to the initial dosage in the course of the treatment. Such high initial dose may also be referred to as “loading dose”. Initiating the treatment of a patient with a loading dose has the effect that a therapeutically effective concentration of the active agent in the patient is reached rapidly, which is particularly useful for active agents that have long elimination half-lives.

[0213] In some embodiments, the subject is treated with 1 dosage of about 3, about 6 or about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle, with the provision that the treatment of the subject comprises at most 2 treatment cycles with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle. This means that the treatment comprises at most 2 treatment cycles with a dosage of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle. The remaining dosages of the anti-CTLA4 antibody or fragment thereof administered in the course of the latter treatment are then selected from about 3 or about 6 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof.

[0214] In some embodiments, the subject is treated with at least 2 dosages of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof in at least 2 different treatment cycles and one or more dosages of about 3 mg / kg body weight or about 6 mg / kg body weight of the

[0215] [P2025W001] / / 241356WO anti-CTLA4 antibody or fragment thereof per treatment cycle in one or more additional treatment cycles.

[0216] In some embodiments, the subject is treated by administering a first dose of about 10 mg / kg body weight, a second dose of about 10 mg / kg body weight, and one or more subsequent doses of about 1-6 mg / kg body weight. In some embodiments, the subject is treated by administering a first dose of about 10 mg / kg body weight, a second dose of about 10 mg / kg body weight, and one or more subsequent doses of about 6 mg / kg body weight.

[0217] In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle and a dosage of 3 mg / kg body weight of the anti-CTLA4 antibody in each subsequent treatment cycle. In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 2ndtreatment cycle, and a dosage of 3 mg / kg body weight of the anti-CTLA4 antibody in each subsequent treatment cycle. In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 2ndtreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 3rdtreatment cycle, and a dosage of 3 mg / kg body weight of the anti-CTLA4 antibody in each subsequent treatment cycle. In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti- CTLA4 antibody in a 1sttreatment cycle, a dosage of 10 mg / kg body weight of the anti- CTLA4 antibody in a 2ndtreatment cycle, a dosage of 10 mg / kg body weight of the anti- CTLA4 antibody in a 3rdtreatment cycle, a dosage of 10 mg / kg body weight of the anti- CTLA4 antibody in a 4thtreatment cycle, and a dosage of 3 mg / kg body weight of the anti- CTLA4 antibody in each subsequent treatment cycle.

[0218] In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle and a dosage of 6 mg / kg body weight of the anti-CTLA4 antibody in subsequent treatment cycles. In some embodiments, the subject is

[0219] [P2025W001] / / 241356WO treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 2ndtreatment cycle, and a dosage of 6 mg / kg body weight of the anti-CTLA4 antibody in each subsequent treatment cycle. In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 2ndtreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 3rdtreatment cycle, and a dosage of 6 mg / kg body weight of the anti-CTLA4 antibody in each subsequent treatment cycle. In some embodiments, the subject is treated with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 1sttreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 2ndtreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 3rdtreatment cycle, a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody in a 4thtreatment cycle, and a dosage of 6 mg / kg body weight of the anti-CTLA4 antibody in each subsequent treatment cycle.

[0220] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered on the first day of each treatment cycle.

[0221] The anti-CTLA4 antibody or fragment thereof can be administered via any suitable route of administration, e.g., parenteral route of administration.

[0222] The anti-CTLA4 antibody or fragment thereof can be administered using any suitable method, such as by oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The suitable route and method of administration may vary depending on a number of factors such as the specific therapeutic agent being used, the rate of absorption desired, specific formulation or dosage form used, type or severity of the disorder being treated, the specific site of action, and conditions of the subject, and can be readily selected by a person skilled in the art. Administration can be systemic or local.

[0223] [P2025W001] / / 241356WO In some embodiments, the anti-CTLA4 antibody or fragment thereof may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the anti-CTLA4 antibody or fragment thereof is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. In some embodiments, the anti-CTLA4 antibody or fragment thereof is formulated for systemic administration. In some embodiments, the systemic administration is by intravenous administration. In some embodiments, the anti-CTLA4 or fragment thereof is formulated for intravenous administration.

[0224] In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered intravenously. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered via an intravenous injection or an intravenous infusion. In some embodiments, the anti-CTLA4 antibody or fragment thereof is administered via an intravenous infusion.

[0225] In some embodiments, the anti-CTLA4 antibody of fragment thereof can be administered over 5-60 minutes, preferably over 10-60 minutes, more preferably over 20-60 minutes, even more preferably over 30-60 minutes, most preferably over 40-60 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 10 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 20 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 30 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 40 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 50 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 60 minutes. In some embodiments, the anti-CTLA4 antibody of fragment thereof is administered over 60 minutes via an intravenous infusion.

[0226] In some embodiments, the anti-CTLA4 antibody for use in a method of treating NSCLC in a subject in need thereof is administered to the subject for up to 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle,

[0227] [P2025W001] / / 241356WO wherein the NSCLC has a squamous histology, and wherein the anti-CTLA4 antibody comprises: a) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

[0228] In some embodiments, the anti-CTLA4 antibody for use in a method of treating NSCLC in a subject in need thereof is administered to the subject for 17 consecutive treatment cycles with 1 dosage of 3 mg / kg body weight of the anti-CTLA4 antibody per treatment cycle, wherein the NSCLC has a squamous histology, and wherein the anti-CTLA4 antibody comprises: a) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

[0229] In some embodiments, the anti-CTLA4 antibody for use in a method of treating NSCLC in a subject in need thereof is administered to the subject in a first dose of 10 mg / kg body weight,

[0230] [P2025W001] / / 241356WO a second dose of 10 mg / kg body weight, and one or more subsequent doses of 6 mg / kg body weight, wherein the NSCLC has a squamous histology, and wherein the anti-CTLA4 antibody comprises: a) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

[0231] In some embodiments, anti-CTLA4 antibody for use in a method of treating NSCLC in a subject in need thereof is administered to the subject in a first dose of 10 mg / kg body weight, a second dose of 10 mg / kg body weight, and in 15 subsequent doses of 6 mg / kg body weight, wherein the NSCLC has a squamous histology, and wherein the anti-CTLA4 antibody comprises: a) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

[0232] [P2025W001] / / 241356WO In some such embodiments, the treatment cycle has about 18 to about 24 days.

[0233] In some embodiments, the anti-CTLA4 antibody or fragment thereof is formulated with one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0234] Suitable carriers include, for example, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, trehalose dihydrate, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxy ethylene / polyoxy-propylene copolymers. Suitable carriers for parenteral administration are preferably isotonic to the blood of the recipient. Non-limiting examples of suitable diluents include ethanol, glycerol, dextrose, and water. Examples of excipients, include without limitation carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0235] In some embodiments, the subject has not previously been treated for cancer, i.e., it is treatment naive. In some embodiments, the subject has previously not been treated for NSCLC with squamous histology.

[0236] In some embodiments, the subject has been previously treated for cancer. In some embodiments, the subject had at least one previous immune checkpoint inhibitor treatment and / or at least one previous chemotherapy treatment. In some embodiments, the subject had at least one previous immune checkpoint inhibitor treatment or at least one previous chemotherapy treatment. In some embodiments, the subject had at least one previous immune checkpoint inhibitor treatment and at least one previous chemotherapy treatment.

[0237] In some embodiments, the at least one previous chemotherapy treatment comprises or is a platinum-comprising chemotherapy. In some embodiments, the platinum-containing chemotherapy comprises or is selected from satraplatin, cisplatin, carboplatin, nedaplatin, lobaplatin, oxaliplatin or combinations thereof.

[0238] [P2025W001] / / 241356WO In some embodiments, the at least one previous chemotherapy treatment comprise or consists of treatment with lurbinectedin, topotecan, paclitaxel, nanoparticle albumin-bound paclitaxel (nab-paclitaxel), pemetrexed, 5-fluoruracil, irinotecan, etoposide, gemcitabine, docetaxel or combinations thereof.

[0239] In some embodiments, the subject has previously not been treated with docetaxel, i.e., it is treatment naive for docetaxel.

[0240] In some embodiments, the at least one previous immune checkpoint inhibitor treatment comprises or is a PD-1 and / or PD-L1 immune checkpoint inhibitor-based treatment. PD-1 and PD-L1 immune checkpoint inhibitors belong to the class of immune checkpoint anti-cancer drugs and target the “Programmed Death- 1 (PD-1)” and “Programmed Death Ligand- 1 (PD- Ll)" receptors. PD-1 and PD-L1 immune checkpoint inhibitors are usually anti -PD-1 and anti-PD-Ll antibodies. Non limiting examples of anti-PD-1 antibodies are pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, pidilizumab (U.S. Pat. No. 7,332,582), AMP-514 (Medlmmune LLC, Gaithersburg, MD), PDR001 (U.S. Pat. No. 9,683,048), BGB-A317 (U.S. Pat. No. 8,735,553), and MGA012 (MacroGenics, Rockville, MD). Non-limiting examples of anti-PD-Ll antibodies are atezolizumab, avelumab, and durvalumab. The phrase “a PD-1 and / or PD-L1 immune checkpoint inhibitor-based treatment” as used herein means that the subject has been treated at least once with a PD-1 immune checkpoint inhibitor and / or a PD-L1 immune checkpoint inhibitor. In some embodiments, the subject has been treated at least once with an anti-PD-1 antibody and / or an anti-PD-Ll antibody. In some embodiments, the subject has been treated at least once with an anti-PD-1 antibody. In some embodiments, the subject has been treated at least once with an anti-PD-Ll antibody. In some embodiments, the subject has been treated at least once with an anti-PD-1 antibody and an anti-PD-Ll antibody.

[0241] In some embodiments, the NSCLC with squamous histology is resistant to treatment with immune checkpoint inhibitors. In some embodiments, the NSCLC with squamous histology is resistant to treatment with PD-1 immune checkpoint inhibitors and / or PD-L1 immune

[0242] [P2025W001] / / 241356WO checkpoint inhibitors. In some embodiments, the NSCLC with squamous histology is resistant to treatment with PD-1 immune checkpoint inhibitors. In some embodiments, the NSCLC with squamous histology is resistant to treatment with PD-L1 immune checkpoint inhibitors. In some embodiments, the NSCLC with squamous histology is resistant to treatment with PD- 1 and PD-L1 immune checkpoint inhibitors. In some embodiments, the NSCLC with squamous histology is resistant to treatment with anti-PD-1 antibodies and / or anti-PD-Ll antibodies. In some embodiments, the NSCLC with squamous histology is resistant to treatment with anti-PD-1 antibodies. In some embodiments, the NSCLC with squamous histology is resistant to treatment with anti-PD-Ll antibodies. In some embodiments, the NSCLC with squamous histology is resistant to treatment with anti-PD-1 antibodies and anti- PD-Ll antibodies.

[0243] In some embodiments, the subject afflicted with NSCLC with squamous histology and to be treated with the anti-CTLA4 antibody as disclosed herein has a primary or acquired resistance to treatment with immune checkpoint inhibitors, particularly to treatment with anti-PDl and / or anti-PD-Ll antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has a primary resistance to treatment with anti-PDl antibodies and / or anti-PD-Ll antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has a primary resistance to treatment with anti-PDl antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has a primary resistance to treatment with anti-PD-Ll antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has a primary resistance to treatment with anti-PDl antibodies and anti-PD-Ll antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has an acquired resistance to treatment with anti-PDl antibodies and / or anti-PD-Ll antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has an acquired resistance to treatment with anti-PDl antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has an acquired resistance to treatment with anti-PD-Ll antibodies. In some embodiments, the subject afflicted with NSCLC with squamous histology has an acquired resistance to treatment with anti-PDl antibodies and anti-PD-Ll antibodies.

[0244] [P2025W001] / / 241356WO In some embodiments, the NSCLC with squamous histology is negative for targetable NSCLC driver mutations. Non-limiting examples of NSCLC driver mutations are Epidermal Growth Factor Receptor (EGFR) gene mutations and anaplastic lymphoma kinase (ALK) gene mutations (Luo et al., Transl Respir Med. 2013, 1(1):6). In some embodiments, the NSCLC with squamous histology is negative for EGFR and / or ALK mutations. In some embodiments, the NSCLC with squamous histology is negative for EGFR mutations. In some embodiments, the NSCLC with squamous histology is negative for ALK mutations. In some embodiments, the NSCLC with squamous histology is negative for EGFR and ALK mutations. Without wishing to be bound by theory, it is contemplated herein that the treatment with anti-CTLA antibodies as disclosed herein is particularly effective for the treatment of squamous NSCLC patients without targetable NSCLC driver mutations. In some embodiments, the NSCLC with squamous histology can be positive for targetable NSCLC driver mutations. In some embodiments, the NSCLC with squamous histology is positive for Kirsten rat sarcoma (KRAS) mutations.

[0245] In some embodiments, the overall survival in a subject afflicted with NSCLC with squamous histology is increased compared to a monotherapy with a chemotherapy agent. In some embodiments, the overall survival in a subject afflicted with NSCLC with squamous histology is increased compared to a monotherapy with docetaxel.

[0246] In some embodiments, the median progression-free survival in a subject afflicted with NSCLC with squamous histology is increased compared to a monotherapy with a chemotherapy agent. In some embodiments, the median progression-free survival in a subject afflicted with NSCLC with squamous histology is increased compared to a monotherapy with docetaxel.

[0247] In some embodiments, the NSCLC with squamous histology has locally advanced or metastasized. The skilled person knows how to determine whether the NSCLC with squamous histology has locally advanced or metastasized, e.g., by using X-ray methods, ultrasound methods, imaging methods (e.g., computer tomography (CT), positron emission

[0248] [P2025W001] / / 241356WO tomography-computed tomography (PET-CT)), and / or the parameters defined in Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1.

[0249] The method of treating non-small cell lung cancer

[0250] The invention also provides a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the method comprises administering an anti-CTLA4 antibody or fragment thereof to the subject, and wherein the NSCLC has a squamous histology.

[0251] The embodiments disclosed herein for the method of treatment comprising the anti-CTLA4 antibody or fragment thereof can be used in the method of treating as disclosed herein. Features described herein in more detail in connection with the “anti-CTLA4 antibody or fragment thereof for use in a method of treating” embodiments equally apply to the corresponding method of treating embodiments.

[0252] The composition comprising the anti-CTLA4 antibody

[0253] In invention further provides a composition comprising an anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology.

[0254] In some embodiments, the composition is a pharmaceutical composition.

[0255] In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0256] In some embodiments, the anti-CTLA-4 antibody has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL. In some embodiments, the anti-CTLA- 4 antibody has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL from a formulation containing 30-60 mg / mL anti-CTLA-4 antibody or

[0257] [P2025W001] / / 241356WO fragment thereof, 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0.

[0258] In some embodiments, the anti-CTLA4 antibody of fragment thereof disclosed herein is formulated in 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0, wherein in some embodiments the concentration of the anti-CTLA4 antibody fragment thereof can be in a certain range, e.g. 2.5 to 100 mg / mL, 5 to 90 mg / mL, 10 to 80 mg / mL, 15 to 80 mg / mL, 20 to 70 mg / mL, 25 to 65 mg / mL, or 30 to 60 mg / mL. In some embodiments, the anti-CTLA4 antibody of fragment thereof disclosed herein is formulated in 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0, wherein the concentration of the anti-CTLA4 fragment thereof is 30 mg / mL. In some embodiments, the anti-CTLA4 antibody of fragment thereof disclosed herein is formulated in 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0, wherein the concentration of the anti-CTLA4 fragment thereof is 60 mg / mL.

[0259] In some embodiments, the anti-CTLA4 antibody or fragment thereof has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL from a formulation comprising 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0, wherein in some embodiments the concentration of the anti-CTLA4 antibody or fragment thereof can be in a certain range e.g. 2.5 to 100 mg / mL, 5 to 90 mg / mL, 10 to 80 mg / mL, 15 to 80 mg / mL, 20 to 70 mg / mL, 25 to 65 mg / mL, or 30 to 60 mg / mL. In some embodiments, the anti-CTLA4 antibody or fragment thereof has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL from a formulation comprising 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, 0.06% (w / v) PS80 at pH 6.0, and 30 mg / mL of the anti-CTLA4 antibody of fragment thereof. In some embodiments, the anti-CTLA4 antibody or fragment thereof has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL from a formulation comprising 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, 0.06% (w / v) PS80 at pH 6.0, and 60 mg / mL of the anti-CTLA4 antibody of fragment thereof.

[0260] [P2025W001] / / 241356WO In some embodiments, the composition comprises: a) the anti-CTLA4 antibody or fragment thereof as disclosed herein, b) about 20 mM histidine buffer, c) about 8.8% (w / v) trehalose dihydrate, and d) about 0.06% (w / v) PS80, at pH 6.0, wherein in some embodiments the concentration of the anti-CTLA-4 antibody can be 2.5 to 100 mg / mL, 5 to 90 mg / mL, 10 to 80 mg / mL, 15 to 80 mg / mL, 20 to 70 mg / mL, 25 to 65 mg / mL, 30 to 60 mg / mL, 30 mg / mL, or 60 mg / mL

[0261] The pH is preferably measured at 22 °C.

[0262] The embodiments disclosed herein for the method of treatment comprising the anti-CTLA4 antibody or fragment thereof can be used in the composition for use as disclosed herein. Features described herein in more detail in connection with the “anti-CTLA4 antibody or fragment thereof for use in a method of treating” embodiments equally apply to the corresponding composition for use embodiments.

[0263] SEQUENCE LISTING

[0264] This application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing file is named 241356US_sequence_listing.xml and 16.1 KB in size.

[0265] SEQ ID NOs: 1-6 are exemplary amino acid sequences of heavy and light chain CDR1-3 sequences of the anti-CTLA4 antibody.

[0266] SEQ ID NOs: 7 and 8 are exemplary amino acid sequences of the heavy and light chain variable domain of the anti-CTLA4 antibody.

[0267] [P2025W001] / / 241356WO SEQ ID NO: 9 is an exemplary amino acid sequence of a mutated Fc region suitable for the anti-CTLA4 antibody.

[0268] SEQ ID NO: 10 and 12 are exemplary amino acid sequences of the full-length heavy and light chain of the anti-CTLA4 antibody.

[0269] SEQ ID NOs: 11 and 13 are exemplary polynucleotide sequences of the full-length heavy and light chain of the anti-CTLA4 antibody.

[0270] SEQ ID NO: 14 is an exemplary amino acid sequence of an unmutated Fc region.

[0271] FIGURE

[0272] Figures 1(a) and 1(b) shows the percent change of the target lesion diameter (tumour size) from the baseline over time in subjects affected with squamous NSCLC and treated with either the anti-CTLA4 antibody or docetaxel. Two dosages of the anti-CTLA4 antibody were administered at 10 mg / kg body weight Q3W followed by 6 mg / kg Q3W. Docetaxel was administered at 75 mg / m2body surface area (PR = partial response, SD = stable disease, PD = progressive disease, NE = not evaluable, NA = not analyzed). Figure 1(a): percent change of the target lesion diameter from the baseline over time in subjects affected with squamous NSCLC and treated with the anti-CTLA4 antibody. Figure 1(b): percent change of the target lesion diameter from the baseline over time in subjects affected with squamous NSCLC and treated with docetaxel.

[0273] Figures 2(a) and (b) - Overall Survival Analysis. Figure 2(a) shows the Kaplan-Meier curve for the Clinical Trial showing the estimated probability of a death event occurring over time for squamous patients (patients suffering from sqNSCLC). Figure 2(b) shows the Kaplan- Meier curve for the Clinical Trial showing the estimated probability of a death event occurring over time for a combined cohort of both squamous and non-squamous patients. For both figures 2(a) and 2(b), the Y-axis represents the survival probability (%) and the X-axis represents the time in months since the patient was randomized and received the first dose of

[0274] [P2025W001] / / 241356WO the anti-CTLA4 antibody (10 mg / kg loading dose followed by 6 mg / kg) or docetaxel (75 mg / m2). The anti-CTLA4 antibody treated patients are shown with a black solid line and the docetaxel patients are shown with a black dashed line.

[0275] Figure 3 - Anti-Tumor Responses Related to the anti-CTLA4 antibody versus docetaxel. Figure 3 is a waterfall plot based on the squamous only (including patients treated with the anti-CTLA4 antibody (10 mg / kg loading dose followed by 6 mg / kg) or docetaxel (75 mg / m2)) cohort showing the best percentage change of target lesion diameter reduction versus the baseline tumor measurement. The Y-axis shows the best percentage change from the baseline diameter in target lesion. The X-axis shows, with horizontal line column, individual patients being treated with the anti-CTLA4 antibody (10 mg / kg loading dose followed by 6 mg / kg) and patients shown as vertical line columns were being treated with docetaxel (75 mg / m2).

[0276] Figure 4 - Progression Free Survival. Figure 4(a) shows the Kaplan-Meier curve for the Clinical Trial showing the estimated probability of a patient not experiencing disease progression over time for squamous patients. Figure 4(b) shows the Kaplan-Meier curve for the Clinical Trial showing the estimated probability of a patient not experiencing disease progression over time for non-squamous patients. For both figures 4(a) and 4(b), the Y-axis represents the survival probability (%) and the X-axis represents the time in months since the patient was randomized and received the first dose of the anti-CTLA4 antibody (10 mg / kg loading dose followed by 6m / kg) or docetaxel (75 mg / m2). The anti-CTLA4 antibody treated patients are shown with a black solid line and the docetaxel patients are shown with a black dashed line.

[0277] EXAMPLE 1

[0278] A clinical study is performed to compare the therapeutic effect of an anti-CTLA4 antibody monotherapy treatment with a monotherapy treatment with the chemotherapy agent docetaxel in locally advanced or metastatic NSCLC with a squamous histology. The NSCLC with

[0279] [P2025W001] / / 241356WO squamous histology is further resistant to the treatment with anti-PD-l / anti-PD-Ll inhibitors and platinum-based chemotherapy and is negative for targetable NSCLC driver mutations (except potential KRAS mutations). The anti-CTLA4 antibody comprises a heavy chain having an amino acid sequence set forth in SEQ ID NO: 10 and a light chain having an amino acid sequence set forth in SEQ ID NO: 12.

[0280] The eligibility criteria for subjects are described in table 2 below.

[0281] Table 2 Eligibility criteria

[0282] [P2025W001] / / 241356WO

[0283] The subjects are subdivided into the following groups:

[0284] • Group A (multiple anti-CTLA4 antibody dosing): the anti-CTLA4 antibody is administered in a Q3 week (21 -day) monotherapy schedule for 17 treatment cycles or up to one year. During the treatment, the anti-CTLA antibody is administered as a single dose of 6 mg / kg body weight but the first and second dosages are increased to 10 mg / kg body weight of the anti-CTLA4 antibody, meaning two dosages of 10 mg / kg body weight are administered Q3W followed by 6 mg / kg Q3W. The anti-CTLA4 antibody is administrated by intravenous infusion in 60 minutes on day 1 of each cycle.

[0285] • Group B (docetaxel): the chemotherapy agent docetaxel is administered in a Q3 week (21 -day) monotherapy schedule for 17 treatment cycles or up to one year. During each treatment cycle, docetaxel is administered at 75 mg / m2body surface area. Docetaxel is administrated by intravenous infusion in 60 minutes on day 1 of each cycle.

[0286] As shown in figures la and lb and figure 3, the anti-CTLA-4 antibody treatment (group A) of squamous NSCLC resulted in an improved decrease of target lesion diameter compared to the docetaxel treatment (group B) of squamous NSCLC. The results shown in figures 1-4 and tables 5-7 surprisingly showed an effective and safe treatment, and an unexpected beneficial effect compared to the docetaxel monotherapy treatment. This demonstrates that the anti- CTLA4 antibody monotherapy treatment is particularly suitable for the treatment of patients with locally advanced or metastasized NSCLC with squamous histology that is resistant to immune checkpoint inhibitor cancer immunotherapy (anti-PD-l / anti-PD-Ll inhibitors) and platinum-based chemotherapy and is negative for targetable NSCLC driver mutations.

[0287] [P2025W001] / / 241356WO EXAMPLE 2

[0288] A clinical study was performed to compare the therapeutic effect of an anti-CTLA4 antibody monotherapy treatment in locally advanced or metastatic NSCLC with a squamous histology (sqNSCLC) or non-squamous histology (non-sqNSCLC). The sqNSCLC and non-sqNSCLC were further resistant to the treatment with anti-PD-l / anti-PD-Ll inhibitors and platinumbased chemotherapy and were negative for targetable NSCLC driver mutations (except potential KRAS mutations). The anti-CTLA4 antibody comprised a heavy chain having an amino acid sequence set forth in SEQ ID NO: 10 and a light chain having an amino acid sequence set forth in SEQ ID NO: 12. The eligibility criteria for subjects are described in table 3 below.

[0289] Table 3 Eligibility criteria

[0290] [P2025W001] / / 241356WO

[0291] The subjects were subdivided into the following groups:

[0292] • Group A (multiple anti-CTLA4 antibody dosing, treatment of sqNSCLC): the anti- CTLA4 antibody is administered in a Q3 week (21 -day) monotherapy schedule for 17 treatment cycles or up to one year. During the treatment, the anti-CTLA antibody is administered as a single dose of 6 mg / kg body weight but the first and second dosages are increased to 10 mg / kg body weight of the anti-CTLA4 antibody, meaning two dosages of 10 mg / kg body weight are administered Q3W followed by 6 mg / kg Q3W. The anti-CTLA4 antibody is administrated by intravenous infusion in 60 minutes on day 1 of each cycle.

[0293] • Group B (multiple anti-CTLA4 antibody dosing, treatment of non-sqNSCLC): the anti-CTLA4 antibody was administered in a Q3 week (21 -day) monotherapy schedule for 17 treatment cycles or up to one year. During the treatment, the anti-CTLA antibody was administered as a single dose of 6 mg / kg body weight but the first and second dosages were increased to 10 mg / kg body weight of the anti-CTLA4 antibody, meaning two dosages of 10 mg / kg body weight were administered Q3W followed by 6 mg / kg Q3W. The anti-CTLA4 antibody was administrated by intravenous infusion in 60 minutes on day 1 of each cycle.

[0294] [P2025W001] / / 241356WO The results of the clinical trial, as shown in table 4, demonstrated an unexpected improvement in overall survival in patients with squamous NSCLC (Group A) compared to patients with non-squamous NSCLC (Group B) when treated with the anti-CTLA4 antibody.

[0295] Table 4 Demographics and OS rates of the anti-CTLA4 antibody monotherapy activity in PD(L)-1 inhibitor resistant NSCLC.

[0296] Demographics and Baseline Characteristics of those patients participating in the Clinical Trial of examples 1 and 2 are shown in table 5 below. The data is displayed in two cohorts: i) squamous patients who received either the anti-CTLA-4 antibody (two loading dosages 10 mg / kg followed by 6 mg / kg) or docetaxel (75 mg / m2); and ii) combined squamous and non- squamous patients who received either the anti-CTLA-4 antibody (two loading dosages 10 mg / kg followed by 6 mg / kg) or docetaxel (75 mg / m2).

[0297] [P2025W001] / / 241356WO Table 5 Demographics and Baseline Characteristics of the patients participating in the Clinical Trial

[0298] [P2025W001] / / 241356WO As shown in figure 2 and table 6, the overall survival (OS) improves upon the anti-CTLA4 antibody treatment compared to the docetaxel treatment (mOS: not evaluable (NE) vs 10.18 months) for sqNSCLC with curves separating starting at 6 months after randomization. Table 6 shows a statistical summary of the results of both figure 2(a) and figure 2(b), including a percentage of overall survival at greater than six (6) months and greater than twelve (12) months since randomization. These data show a hazard ratio for both the squamous and non- squamous patient cohorts.

[0299] Table 6 Overall Survival Analysis In table 7, a summary of the overall response rate (“ORR”) for the various cohorts of patients is shown, namely i) squamous patients treated with the anti-CTLA4 antibody (two loading dosages 10 mg / kg followed by 6 mg / kg); ii) squamous patients treated with docetaxel (75mg / m2); iii) squamous and non-squamous patients treated with the anti-CTLA4 antibody (two loading dosages 10 mg / kg followed by 6 mg / kg); iv) squamous and non-squamous

[0300] [P2025W001] / / 241356WO patients treated with docetaxel (75mg / m2). Each cohort of patients was evaluated by a Blinded independent central review (BICR) analysis or per the Investigator.

[0301] Table 7 Anti-Tumor Responses Related to the anti-CTLA4 antibody Versus Docetaxel

[0302] [P2025W001] / / 241356WO

Claims

C l a i m s1. An anti-CTLA4 antibody or fragment thereof for use in a method of treating nonsmall cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology.

2. The anti-CTLA4 antibody or fragment thereof for use according to claim 1, wherein the anti-CTLA4 antibody or fragment thereof comprises: a) a heavy chain variable region comprising (1) a complementarity-determining region 1 (HCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1, (2) a complementarity-determining region 2 (HCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2, and (3) a complementarity-determining region 3 (HCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3; and b) a light chain variable region comprising (1) a complementarity-determining region 1 (LCDR1) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4, (2) a complementarity-determining region 2 (LCDR2) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 5, and (3) a complementarity-determining region 3 (LCDR3) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 6.

3. The anti-CTLA4 antibody or fragment thereof for use according to claims 1 or 2, wherein the anti-CTLA4 antibody is administered as monotherapy.

4. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-3, wherein the anti-CTLA4 antibody or fragment thereof dosage administered[P2025W001] / / 241356WOranges from about 0.1 mg / kg to about 45 mg / kg body weight, optionally from about 1 mg / kg to about 30 mg / kg body weight, optionally from about 2 mg / kg to about 20 mg / kg body weight.

5. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-4, wherein the anti-CTLA4 antibody or fragment thereof dosage administered is independently selected from about 1 mg / kg body weight, about 3 mg / kg body weight, about 6 mg / kg body weight, about 10 mg / kg body weight, about 12 mg / kg body weight, about 15 mg / kg body weight, and about 20 mg / kg body weight, optionally, wherein the anti-CTLA4 antibody or fragment thereof dosage administered is independently selected from about 3 mg / kg body weight, about 6 mg / kg body weight, and about 10 mg / kg body weight.

6. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-5, wherein a treatment cycle is repeated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.

7. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-6, wherein each treatment cycle has about 18 to about 28 days, optionally about 18 to about 24 days, optionally about 21 days.

8. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-7, wherein the anti-CTLA4 antibody or fragment thereof is administered about every 4 weeks, optionally about every 3 weeks.

9. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-8, wherein the anti-CTLA4 antibody or fragment thereof is administered for 17 treatment cycles or up to 1 year.[P2025W001] / / 241356WO10. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-9, wherein the subject is treated with 1 dosage of the anti-CTLA4 antibody or fragment thereof per treatment cycle.

11. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-10, wherein the subject is treated with at least 1 dosage of about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof, optionally wherein the subject is treated with at least 2 dosages of about 10 mg / kg body weight of the anti- CTLA4 antibody or fragment thereof.

12. The anti-CTLA4 antibody or fragment thereof for use according to claims 1-11, wherein the subject is treated with 1 dosage of about 3, about 6 or about 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle, with the provision that the treatment of the subject comprises at most 2 treatment cycles with a dosage of 10 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle.

13. The anti-CTLA4 antibody or fragment thereof for use according to claim 12, comprising administering a first dose of about 10 mg / kg body weight, a second dose of about 10 mg / kg body weight, and one or more subsequent doses of about 1-6 mg / kg body weight, optionally wherein each subsequent dose is about 6 mg / kg body weight.

14. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-10, wherein the subject is treated with 1 dosage of about 3 mg / kg body weight of the anti-CTLA4 antibody or fragment thereof per treatment cycle.

15. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-14, wherein the anti-CTLA4 antibody or fragment thereof is administered on the first day of each treatment cycle.[P2025W001] / / 241356WO16. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-15, wherein the anti-CTLA4 antibody or fragment thereof is administered intravenously.

17. The anti-CTLA4 antibody or fragment thereof for use according to claim 16, wherein the anti-CTLA4 antibody or fragment thereof is administered via an intravenous injection or an intravenous infusion, optionally an intravenous infusion.

18. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-17, wherein the subject is a human.

19. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-18 wherein the anti-CTLA4 antibody or fragment thereof is formulated with one or more pharmaceutically acceptable carriers, diluents and / or excipients.

20. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-19, wherein the subject has not previously been treated for cancer, optionally wherein the subject has previously not been treated for NSCLC with squamous histology.

21. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-19, wherein the subject has been previously treated for cancer, optionally wherein the subject had at least one previous immune checkpoint inhibitor treatment and / or at least one previous chemotherapy treatment.

22. The anti-CTLA4 antibody or fragment thereof for use according to claim 21, wherein the at least one previous immune checkpoint inhibitor treatment comprises or is a PD- 1 and / or PD-L1 immune checkpoint inhibitor-based treatment.[P2025W001] / / 241356WO23. The anti-CTLA4 antibody or fragment thereof for use according to claim 21 or 22, wherein the at least one previous chemotherapy treatment comprises or is a platinum- comprising chemotherapy.

24. The anti-CTLA4 antibody or fragment thereof for use according to claim 23, wherein the platinum-containing chemotherapy comprises or is selected from satraplatin, cisplatin, carboplatin, nedaplatin, lobaplatin, oxaliplatin or combinations thereof.

25. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-24, wherein the overall survival in said subject is increased compared to a monotherapy with a chemotherapy agent.

26. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-25, wherein the median progression-free survival in said subject is increased compared to a monotherapy with a chemotherapy agent.

27. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-26, wherein the NSCLC with squamous histology has locally advanced or metastasized.

28. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-27, wherein the NSCLC with squamous histology is negative for targetable NSCLC driver mutations.

29. The anti-CTLA4 antibody or fragment thereof for use according to claim 28, wherein the NSCLC with squamous histology is negative for EGFR and / or ALK mutations.

30. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-27, wherein the NSCLC with squamous histology is positive for KRAS mutations.[P2025W001] / / 241356WO31. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-30, wherein the wherein the anti-CTLA4 antibody or fragment thereof comprises: a) a heavy chain variable domain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7; and b) a light chain variable domain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8.

32. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1- 31, wherein the anti-CTLA4 antibody or fragment thereof comprises a constant region optionally derived from a human antibody, optionally the constant region is selected from the constant region of human IgGl, IgG2, IgG3 or IgG4.

33. The anti-CTLA4 antibody or fragment thereof for use according to claim 32, wherein the constant region is human IgGl constant region.

34. The anti-CTLA4 antibody or fragment thereof for use according to claim 33, wherein the anti-CTLA4 antibody or fragment thereof comprises an IgGl Fc, optionally comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.

35. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-34, wherein the anti-CTLA4 antibody comprises:[P2025W001] / / 241356WOa) a heavy chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10; and b) a light chain comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence comprising at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12.

36. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-35, wherein the anti-CTLA4 antibody or fragment thereof is capable of binding to human CTLA4.

37. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-36, wherein the anti-CTLA antibody or fragment thereof is a humanized anti- CTLA4 antibody or fragment thereof.

38. The anti-CTLA4 antibody or fragment thereof for use according to any one of claims 1-31, 36, or 37, wherein the anti-CTLA antibody fragment is an antigen-binding fragment or a variant thereof.

39. The anti-CTLA4 antibody or fragment thereof for use according to claim 38, wherein the antigen binding fragment or variant thereof is selected from the group consisting of a Fab, a Fab’, a F(ab’)2, a scFV, a rlgG, a diabody, a triabody, a minibody, and a single-domain antibody (sdAB), and variants thereof.

40. A method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the method comprises administering an anti-CTLA4 antibody or fragment thereof to the subject, and wherein the NSCLC has a squamous histology.[P2025W001] / / 241356WO41. A composition comprising an anti-CTLA4 antibody or fragment thereof for use in a method of treating non-small cell lung cancer (NSCLC) in a subject in need thereof, wherein the NSCLC has a squamous histology.

42. The composition according to claim 41, wherein the composition is a pharmaceutical composition.

43. The pharmaceutical composition according to claim 42, comprising one or more pharmaceutically acceptable carriers, diluents and / or excipients.

44. The pharmaceutical composition according to any one of claims 42-43, wherein the anti-CTLA-4 antibody has been diluted with 5% Dextrose solution to a final concentration of about 1 to about 12 mg / mL, from a formulation containing 30-60 mg / mL anti-CTLA-4 antibody or fragment thereof, 20 mM histidine buffer, 8.8% (w / v) trehalose dihydrate, and 0.06% (w / v) PS80 at pH 6.0.[P2025W001] / / 241356WO

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