Methods and pharmaceutical compositions for enhancing CD8 positive t cell-dependent immune responses in patients suffering from cancer

Abstract

To provide methods and pharmaceutical compositions for enhancing CD8-positive T cell-dependent immune responses in patients suffering from cancer.SOLUTION: There is provided a method for increasing an amount of tumor-infiltrating CD8 positive T cells in a patient afflicted with cancer comprising administering a therapeutically effective amount of a NRP-1 inhibitor. Also there is provided a method for treating cancer in a patient in need thereof, comprising administering a therapeutically effective combination of an immune checkpoint inhibitor and a NRP-1 inhibitor thereby enhancing therapeutic efficacy relative to the administration of the immune checkpoint inhibitor alone.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field of the invention: The present invention relates to methods and pharmaceutical compositions for enhancing CD8-positive T cell-dependent immune responses in patients suffering from cancer. [Background technology]

[0002] Background of the invention: It was first proposed over 100 years ago that the immune system can detect and eliminate cancer. Since then, T cells reactive against tumor-associated antigens have been detected in the blood of patients with many different types of cancer, suggesting a role for the immune system in fighting cancer. Innate and adaptive immunity maintain effector cells, such as lymphocytes and natural killer cells, that distinguish normal cells from "modified" cells, as in the case of tumor cells. However, in most cases, tumor cells are able to evade immune recognition and destruction. Although there are many tumor evasion mechanisms, the immunosuppressive effect of co-inhibitory molecules has emerged in the last decade as the most attractive mechanism for imagining novel cancer treatments. Lymphocyte activation is indeed regulated by both co-stimulatory and co-inhibitory molecules, belonging to the B7 / CD28 superfamily (also known as the immunoglobulin (Ig) superfamily) and the TNF (tumor necrosis factor) / TNFR (tumor necrosis factor receptor) superfamily. The balance between these signals determines lymphocyte activation and, consequently, regulates the immune response. These costimulatory and co-inhibitory molecules are called "immune checkpoints". The immune checkpoint that has attracted the most attention in recent years is programmed cell death protein 1 (PD-1). Monoclonal antibodies that inhibit PD-1, such as nivolumab and pembrolizumab, have indeed demonstrated significant efficacy, have already been approved, and are expected to become blockbusters in the future. However, despite the considerable progress proposed by these drugs, some patients do not respond, and therefore, in order to provide novel therapeutic options, it is necessary to identify the mechanisms of said resistance.

[0003] Neurofilin-1 (NRP-1) is a transmembrane glycoprotein that acts as a coreceptor for various members of the vascular endothelial growth factor (VEGF) family. Its ability to bind and regulate the activity of many other extracellular ligands, such as class 3 semaphorins, transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (PDGF), suggests the involvement of NRP-1 in various physiological and pathological processes. Indeed, this coreceptor is involved in axon guidance, angiogenesis, and tumor progression. Although we and others have demonstrated the involvement of Nrp1 in both innate and adaptive immunity, the impact of NRP-1 in antitumor cytotoxic T cells has not been investigated so far. Summary of the Invention [Problem to be solved by the invention]

[0004] Summary of the invention: As defined by the claims, the present invention relates to methods and pharmaceutical compositions for enhancing CD8 positive T cell dependent immune responses in patients suffering from cancer. [Means for solving the problem]

[0005] Detailed description of the invention: The present inventors have now demonstrated that NRP-1 is expressed by cytotoxic T cells (CD8 positive T cells or cytotoxic T cells), colocalizes with PD-1, and forms a complex with PD-1 on the cells. Deletion of NRP-1 expression on cytotoxic T cells resulted in a more pronounced antitumor response in animals. Furthermore, the combination of deleting NRP1 on CD8 positive T cells and anti-PD1 inhibitors in animals resulted in a synergistic antitumor effect. Finally, computational analysis shows that reduced expression of NRP-1 in patients is associated with better responses in patients treated with anti-PD1 inhibitors. [Brief description of the drawings]

[0006] [Figure 1] Expression of Nrp1 in CD8-positive T cells of tumor-infiltrating tetramer H2kb / SIINFEKL mice. B16F10-ovalbumin tumor cells were subcutaneously injected into the right flank of wild-type C57BL / 6 mice. 7 and 14 days after tumor injection, mice were subcutaneously immunized with a combination of polyIC / ovalbumin. Tumors were harvested 21 days after injection, and tumor-infiltrating CD8-positive T cells were stained using tetramer H2kb-SIINFEKL, anti-CD8, and anti-Nrp1 antibodies and analyzed by flow cytometry. Data are presented in flow cytometry graphs with anti-CD8 and tetramer H2kb-ovalbumin. Tetramer H2kb-ovalbumin positive populations are presented in histograms with respect to Nrp1 expression normalized to the mode. Data are representative of three independent experiments. [Diagram 2] Expression profile of NRP1 in H2-Db GP33-specific CD8+ T cells following in vivo infection of mice with Lymphocytic Choriomeningitis Virus (LCMV) Armstrong strain (n=16), LCMV clone 13 strain (n=16), or control naive CD44lowCD8+ T cells (n=4) on days 6, 8, 15, and 30. Data for transcriptomics analysis were available from Doering et al. Immunity, 2012. P values ​​were determined by two-dimensional ANOVA (p=0.0008). [Diagram 3] Tracking B16F10 tumor volume in an anti-tumor immune response model. CD8-positive Nrp1 knockout (KO) or CD8CRE (wild type) mice received 1 million B16F10 in the right flank on day 0, followed by immunization with PolyIC / Ovalbumin on days 7 and 14, with 40 μg PolyIC and 400 μg ovalbumin per mouse injected subcutaneously. Data are presented as the mean tumor volume + / - standard error of the mean on days 0, 8, 11, 14, 18, and 21 after injection. Data are representative of three independent experiments. [Figure 4]Percentage of tetramer / PE-H2Kb ovalbumin CD8 positive tumor infiltrating lymphocytes in B16-ovalbumin tumors of four different mouse groups assessed by flow cytometry 14 days after immunization from CD8Nrp1 knockout (KO) and control (wild type, WT) mice immunized or not (control) with ovalbumin and Poly IC. Data are presented as the mean ± standard error of the mean percentage of CD8 positive tumor infiltrating lymphocytes tetramer positive. P values ​​were determined by Student's T test. **p<0.01, *p<0.05. Data are representative of three independent experiments. [Diagram 5] Quantification by ImageStream of NRP1 expression (mean pixel intensity / MPI) in an allogeneic synapse model between activated CD8+ T cells and A20 cells labeled with cell tracer violet. NRP1 expression was analyzed in activated CD8+ T cells at the synaptic junction (zone highly labeled with phalloidin). Data are presented as mean MPI ± standard error of the mean. P-values ​​(p<0.0001) were determined by Wilcoxon signed rank test. Data are representative of four independent experiments derived from two synapse models. [Figure 6] PD1 is recruited into the synapse between activated CD8+ T cells and tumor cells. Imagestream analysis of PD1 expression (mean pixel intensity / MPI) in a model of synapse between activated CD8+ T cells and allogeneic A20 tumor cells: PD1 expression was analyzed in phalloidin-high areas between activated CD8+ T cells and tumor cells (A20). Data are presented as mean MPI ± standard error of the mean. P values ​​were determined by Student's T-test. Data are representative of five experiments. [Figure 7] Flow cytometric analysis of NRP1 and PD1 expression in human CD8-positive tumor-infiltrating lymphocytes. Data are representative of three independent experiments in human uterine, renal, and ovarian cancer. [Figure 8]Quantification by ImageStream of PD1 expression (MPI) at synaptic junctions (highly phalloidin labeled zones) between activated CD8+ T cells from CD8Nrp1 knockout mice (KO) or controls (WT) and allogeneic A20 tumor cells. Data are presented as mean MPI ± standard error of the mean. P values ​​(p<0.0001) were determined by Mann-Whitney test. Data are representative of two independent experiments. [Figure 9] Quantification by ImageStream of the amount of phosphorylated ZAP70 (mean pixel intensity / MPI) at synaptic junctions (highly phalloidin labeled zones) between activated CD8+ T cells from CD8Nrp1 knockout (KO) or control (WT) mice and A20 tumor cells labeled with cell tracer violet. Data are presented as mean MPI ± standard error of the mean. P values ​​(p<0.0001) were determined by Mann-Whitney test. Data are representative of three independent experiments. [Figure 10] Flow cytometric analysis of phosphorylated ZAP70 in human PD1+CD8+ tumor-infiltrating lymphocytes according to NRP1 expression. Data are representative of one experiment in human uterine carcinoma. [Figure 11] Flow cytometric analysis of CD25 expression on CD8+ T cells from patients with NRP1 haploinsufficiency (patients) or controls (N=5) in response to concentrations of Staphylococcal enterotoxin B (SEB) superantigen (0, 1, 10, or 100 ng / mL) in the presence or absence of anti-PD1 antibody. Activation was performed for 72 hours. Data are presented as mean % of CD25 expression ± standard error of the mean. Human anti-PD1 antibody (pembrolizumab, Merck). [Figure 12]Flow cytometric analysis of the proportion of dividing CD8+ T cells from patients with NRP1 haploinsufficiency (patients) or controls (N=5) in response to concentrations of Staphylococcal enterotoxin B superantigen (0, 1, 10, or 100 ng / mL) in the presence or absence of anti-PD1 antibody. Activation was performed for 72 hours. Data are presented as mean ± standard error of the mean. Human anti-PD1 antibody (pembrolizumab, Merck). [Figure 13] CD8Nrp1 knockout (KO) and control (WT) mice were pre-immunized with ovalbumin and Poly IC and treated or not with anti-PD1 antibody in vivo. Overall survival was assessed up to day 50 post-immunization. Data are presented as mean ± standard error of the mean and as Kaplan-Meier curves. P values ​​were determined by log-rank test. Data are representative of 5 experiments. Mouse anti-PD1 antibody (J43 clone) from BioXcells. [Figure 14] Analysis of overall survival of metastatic melanoma patients treated with anti-PD1 antibodies according to the expression of NRP1 RNA (low or high expression) assessed in the tumor before treatment with anti-PD1 antibodies. Data for transcriptome analysis of metastatic melanoma tumors were available from Hugo et al. Cell, 2016. Data are presented as Kaplan-Meier curves. P value (p=0.03) was determined by log-rank test (n=25 patients). [Figure 15] Analysis of recurrence-free survival in metastatic melanoma patients treated with anti-PD-1 antibodies and achieving at least a partial response according to the expression of NRP1 (NRP1- / low compared with NRP1+ / high) in CD8-positive tumor-infiltrating lymphocytes, assessed by immunohistochemistry before starting treatment. NRP1 expression was evaluated by performing a blinded analysis. Data are presented as Kaplan-Meier curves. P value (P=0.042) was determined by the log-rank test (n=15 patients). [Figure 16]Imagestream quantification of the amount of phosphorylated ZAP70 (mean pixel intensity / MPI) in human activated CD8+ T cells at synapses containing tumor cells (Raji cells). Data are presented as mean MPI ± standard error of the mean. P values ​​were determined by Mann-Whitney test. Human anti-NRP1 antibody (AF3870, R&D Systems), human anti-PD-1 antibody (pembrolizumab, Merck). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Key definitions: The term "T cells" as used herein has its general meaning in the art and refers to an important component of the immune system that plays a central role in cell-mediated immunity. T cells are known as conventional lymphocytes because they recognize antigens using their TCR (T cell receptor for antigens) by presentation or restriction by major histocompatibility complex molecules. There are several subsets of T cells, each with distinct functions, such as CD8+ T cells, CD4+ T cells, γδ T cells, and regulatory T cells (Tregs).

[0008] The term "CD8 positive T cells" as used herein has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8 positive T cells" are also called cytotoxic T lymphocytes (CTLs), T killer cells, cytotoxic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is the recognition element associated with major histocompatibility complex class I restricted interactions. The term "tumor infiltrating CD8 positive T cells" as used herein refers to the pool of CD8 positive T cells of a patient that have left the bloodstream and migrated into the tumor.

[0009] As used herein, the term "CD4 positive T cells" (also called helper T cells or TH cells) refers to T cells that express the CD4 glycoprotein on their surface and assist other white blood cells in immunological processes, including maturation of B cells to plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD4 positive T cells become activated when they are presented with peptide antigens expressed on the surface of antigen-presenting cells (APCs) by MHC class II molecules. Once activated, they divide rapidly and secrete cytokines that regulate or assist vigorous immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, TFH (follicular helper T cells), or Treg (regulatory T cells), which secrete different cytokines to promote different kinds of immune responses. Signaling from antigen-presenting cells directs T cells toward specific subtypes. In addition to CD4, surface biomarkers of helper T cells known in the art include CXCR3 (Th1), CCR4, Crth2 (Th2), CCR6 (Th17), CXCR5 (Tfh) and expression of subtype-specific cytokines and transcription factors, including T-bet, GATA3, EOMES, RORγT, BCL6, and FoxP3.

[0010] The term "gamma delta T cells" as used herein has its general meaning in the art. Gamma delta T cells usually account for 1-5% of peripheral blood lymphocytes in healthy individuals (humans, monkeys). They are involved in the initiation of protective immune responses, and they have been shown to recognize their antigenic ligands by direct interaction with the antigen, without any presentation by MHC molecules of antigen-presenting cells. Gamma 9 delta 2 T cells (sometimes also called gamma 2 delta 2 T cells) are gamma delta T cells that have a T cell receptor with variable domains Vγ9 and Vδ2. They form the majority of gamma delta T cells in human blood. When activated, gamma delta T cells exert a potent, non-MHC-restricted cytotoxic activity and are particularly effective in killing various types of cells, especially pathogenic cells. These may be cells infected with viruses (Poccia et al., J. Leukocyte Biology, 1997, 62: 1-5) or with other intracellular parasites, such as mycobacteria (Constant et al., Infection and Immunity, December 1995, vol. 63, no. 12: 4628-4633) or protozoa (Behr et al., Infection and Immunity, 1996, vol. 64, no. 8: 2892-2896). They may also be cancer cells (Poccia et al., J. Immunol., 159: 6009-6015; Fournie and Bonneville, Res. Immunol., 66th Forum in Immunology, 147: 338-347). Therefore, being able to modulate the activity of such cells in vitro, ex vivo, or in vivo provides novel and effective therapeutic approaches in the treatment of a variety of pathologies such as infectious diseases (especially viral or parasitic diseases), cancer, allergies, and even autoimmune and / or inflammatory disorders.

[0011] As used herein, the term "CAR-T cells" refers to T lymphocytes genetically engineered to express a chimeric antigen receptor (CAR). The definition of CAR T cells encompasses all classes and subclasses of T lymphocytes, including CD4+ T cells, CD8+ T cells, gamma delta T cells, and effector T cells, memory T cells, regulatory T cells, etc. Genetically modified T lymphocytes may be "derived from" or "obtained from" a subject who will be treated with the genetically modified T cells, or they may be "derived from" or "obtained from" a different subject.

[0012] The term "chimeric antigen receptor" or alternatively "CAR" as used herein refers to a set of polypeptides, typically two sets of polypeptides in the simplest embodiment, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and the generation of an intracellular signal. In some embodiments, the CAR comprises at least one extracellular antigen binding domain, one transmembrane domain, and an intracytoplasmic signaling domain (also referred to herein as "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides comprises a dimerization switch, which, in the presence of a dimerization molecule, allows the polypeptides to bind to each other, e.g., an antigen binding domain to be linked to the intracellular signaling domain. In some embodiments, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In some embodiments, the intracytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and one functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and one functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, where the leader sequence is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. In certain aspects, the CAR comprises a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody fused to CD3 zeta with a transmembrane domain and an endodomain. In some embodiments, the CAR comprises domains for additional costimulatory signaling, such as CD3 zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules may be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0013] The term "NRP-1" as used herein has its general meaning in the art and refers to neuropilin-1. An exemplary human nucleic acid sequence of NRP-1 is represented by NCBI reference sequence NM_001024628.2, and the human amino acid sequence is represented by NCBI reference sequence NP_001019799.1. In particular, the human amino acid sequence of NRP-1 is represented by SEQ ID NO:1. The basic structure of NRP-1 includes five domains, namely, three extracellular domains (a1a2, b1, b2 and c), one transmembrane domain, and one cytoplasmic domain (SEQ ID NO:1). The a1a2 domain that binds to semaphorin 3A ranges from the amino acid residue at position 1 to the amino acid residue at position 280 of SEQ ID NO:1.

[0014] [ka]

[0015] As used herein, a "functional equivalent of NRP-1" is a polypeptide that can bind to semaphorin 3A, thereby preventing the interaction of NRP-1 with semaphorin 3A. The term "functional equivalent" includes fragments, mutants, and muteins of NRP-1. The term "functionally equivalent" therefore includes any equivalent of NRP-1 obtained by modifying the amino acid sequence, for example by deletion, substitution, or addition of one or more amino acids, so that the protein analogue retains, for example, the ability to bind to semaphorin 3A. Amino acid substitutions can be made, for example, by point mutations in the DNA encoding the amino acid sequence. As used herein, the term "functionally equivalent fragment" can also refer to any fragment or collection of fragments of NRP-1.

[0016] The term "NRP-1 inhibitor" as used herein refers to a compound, substance, or composition that can inhibit the function and / or expression of NRP-1. For example, the inhibitor can inhibit the expression or activity of NRP-1, can regulate or block NRP-1, or can block a signal transduction pathway. In particular, the NRP-1 inhibitor inhibits the interaction between NRP-1 and its counterpart, particularly semaphorin-3A. In particular, the inhibitor of NRP-1 does not inhibit the interaction between NRP-1 and VEGF (vascular endothelial growth factor).

[0017] The term "semaphorin-3A" as used herein has its general meaning in the art and refers to the protein semaphorin-3A encoded by the SEMA3A gene in humans. The term is also known as COLL1, HH16, Hsema-1, Hsema-III, SEMA1, SEMAD, SEMAIII, SEMAL, SemD, and coll-1. The SEMA3A gene is a member of the semaphorin family and encodes a protein having an immunoglobulin-like C2-type (immunoglobulin-like) domain, a PSI domain, and a Sema domain. An exemplary human nucleic acid sequence of NRP-1 is represented by NCBI reference sequence NM_006080.2, and the human amino acid sequence is represented by NCBI reference sequence NP_006071.1. In particular, the human amino acid sequence of semaphorin 3A is represented by SEQ ID NO:2.

[0018] [ka]

[0019] The term "linker" as used herein refers to a sequence of at least one amino acid that connects the polypeptide of the present invention to an immunoglobulin sequence portion. Such linkers may be useful to prevent steric hindrance. In some embodiments, the linker has 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 amino acid residues. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Another example is a poly-alanine linker sequence.

[0020] The term "immune checkpoint protein" as used herein has its general meaning in the art and refers to a molecule expressed by T cells that either up-regulates (stimulatory checkpoint molecule) or down-regulates (inhibitory checkpoint molecule). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The adenosine A2A receptor (A2AR) is considered as a key checkpoint in cancer therapy. This is because the tumor microenvironment has relatively high levels of adenosine, which leads to a negative immune feedback loop through activation of A2AR. B7-H3, also called CD276, was originally understood to be a costimulatory molecule but is now considered to be co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor evasion. B and T lymphocyte attenuator (BTLA), also called CD272, is a ligand for HVEM (herpes virus entry mediator). Cell surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from a naive cell phenotype to an effector cell phenotype, but tumor-specific CD8+ T cells express high levels of BTLA. CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also called CD152, is overexpressed on regulatory T cells and acts to regulate T-cell proliferation. IDO (indoleamine 2,3-dioxygenase) is a tryptophan catabolic enzyme that is a related immunosuppressive enzyme. Another important molecule is TDO (tryptophan 2,3-dioxygenase).IDO is known to suppress T cells and NK cells, generate and activate regulatory T cells and myeloid-derived suppressor cells, and promote tumor angiogenesis. KIR (killer cell immunoglobulin-like receptor) is a receptor for MHC class I molecules on natural killer cells. LAG3 (lymphocyte activation gene-3) acts to suppress immune responses by its effects on regulatory T cells as well as direct effects on CD8+ T cells. TIM-3 (short for T cell immunoglobulin domain and mucin domain 3) is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1 / Tc1 function by triggering cell death upon interaction with its ligand, galectin-9. VISTA (short for V-domain Ig suppressor of T cell activation) is primarily expressed on hematopoietic cells, and thus consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective against a wide range of solid tumors. As used herein, the term "PD-1" has its common meaning in the art and refers to programmed cell death protein 1 (also known as CD279). PD-1 acts as an immune checkpoint and inhibits T-cell activation upon binding to one of its ligands, PD-L1 or PD-L2.

[0021] The term "immune checkpoint inhibitor" as used herein has its general meaning in the art and refers to any compound that inhibits the function of an immune suppressive checkpoint protein. Inhibition includes reduction and complete blocking of function. A preferred immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. Many immune checkpoint inhibitors are known, and alternative immune checkpoint inhibitors can be developed in the (near) future by analogy with these known immune checkpoint protein inhibitors. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules, and small molecules. In particular, the immune checkpoint inhibitor of the present invention is administered to enhance the proliferation, migration, persistence, and / or cytotoxic activity of CD8 positive T cells in a patient, in particular tumor-infiltrating CD8 positive T cells in a patient. The ability of an immune checkpoint inhibitor to enhance the killing activity of CD8 positive T cells can be determined by any assay known in the art. Typically, the assay is an in vitro assay in which CD8 positive T cells are contacted with target cells (e.g., target cells that are recognized and / or lysed by CD8 positive T cells). For example, the immune checkpoint inhibitor of the present invention can be selected for its ability to increase the specific lysis by CD8 positive T cells by more than 20%, preferably at least about 30%, at least about 40%, at least about 50%, or more, obtained by contacting CD8 positive T cells or CD8 positive T cell lines with the immune checkpoint inhibitor of the present invention at the same effector cell:target cell ratio. Examples of protocols for classical cytotoxicity assays are routine. Thus, the expression "enhancing the efficacy of immune checkpoints" refers to the ability of NRP-1 inhibitors to enhance the ability of immune checkpoint inhibitors to enhance the proliferation, migration, persistence, and / or cytotoxicity of CD8 positive T cells.

[0022] Thus, as used herein, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region, and this term includes any antibody-like molecule having an antigen-binding domain, such as Fab', Fab, F(ab')2, single domain antibodies (DAB), tandem antibody dimers, Fv, scFv (single-chain Fv), dsFv (two-chain Fv), ds-scFv, Fd, linear antibodies, miniantibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (bispecific or trispecific scFv-Fab fusions, respectively). ); sc-diabodies; kappa (lambda) bodies (scFv-CL fusions); BiTE antibodies (bispecific T cell inducer antibodies, scFv-scFv attracting T cells); DVD-Ig (dual variable domain antibodies, bispecific format); SIPs (small immune proteins, a type of mini-antibody); SMIPs ("small modular immunopharmaceuticals" scFv-Fc dimers); DARTs (two-chain stabilized diabodies "dual affinity retargeting"); small antibody mimetics containing one or more CDRs, and the like. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies are further described in, among others, EP 404,097 and WO 93 / 11161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies may be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to generate Fab' fragments. Digestion with papain can form Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, domain antibodies, tandem antibodies, ds-scFv, dimers, mini-antibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or chemically synthesized. Techniques for generating antibody fragments are well known and described in the art.For example, Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 each further describe and enable the generation of effective antibody fragments. In some embodiments, the antibodies of the invention are single chain antibodies. As used herein, the term "single domain antibody" has its general meaning in the art and refers to a single heavy chain variable domain of the type of antibody that may be found in camelid mammals that are naturally devoid of light chains. Such single domain antibodies are also referred to as "Nanobodies®". For a general description of (single) domain antibodies, see also the prior art cited above, as well as EP 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388.

[0023] In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains, one variable domain (VL) and one constant domain (CL). The heavy chain contains four domains, one variable domain (VH) and three constant domains (CH1, CH2, CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental transfer, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable portion. The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is made up of residues mainly from the hypervariable or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or may affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the V regions of the heavy and light chains. Framework region (FR) refers to the amino acid sequence inserted between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.Residues in antibody variable domains are usually numbered according to the system devised by Kabat et al., which is set out in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, National Institutes of Health, USA (Kabat et al., 1992, hereafter "Kabat et al."). The Kabat residue designations do not always correspond directly to the linear numbering of the amino acid residues of the sequence of SEQ ID NO:. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering, corresponding to the shortening of or insertion into a structural component, whether a framework region or a complementarity determining region (CDR) of the basic variable domain structure. The correct Kabat numbering of residues for a given antibody can be determined by aligning the homologous residues in the sequence of the antibody with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system. The CDRs for the antibodies described hereinafter have been determined using the CDR discovery algorithm of www.bioinf.org.uk. See the section entitled "How to identify the CDRs by looking at a sequence" in the antibody page.

[0024] The term "single domain antibody" as used herein has its general meaning in the art and refers to a single heavy chain variable domain of an antibody of the type that may be found in camelid mammals that are naturally devoid of light chains. Such single domain antibodies are also "Nanobodies®".

[0025] As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, where the light and heavy chain variable regions are linked sequentially, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, where the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, an scFv as used herein can have a VL variable region and a VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and the scFv can comprise a VL-linker-VH or a VH-linker-VL.

[0026] As used herein, the term "bispecific antibody" refers to an antibody that contains specificity for two target molecules, i.e., an antibody that has specificity for at least two different, typically non-overlapping, epitopes. As used herein, the term "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the full molecule is of human origin or consists of an amino acid sequence that is identical to a human form of an antibody or immunoglobulin.

[0027] "Humanized" as used herein describes antibodies in which some, most, or all of the amino acids outside the CDR regions have been replaced with corresponding amino acids derived from human immunoglobulin molecules.

[0028] The term "cross-competition" as used herein refers to monoclonal antibodies that share the ability to bind to a particular region of an antigen. In the present disclosure, a "cross-competing" monoclonal antibody has the ability to interfere with the binding of another monoclonal antibody to an antigen in a standard competitive binding assay. Such a monoclonal antibody may, by non-limiting theory, bind to the same, related, or nearby (e.g., structurally similar or spatially close) epitope as the antibody it competes with. Cross-competition exists when antibody A reduces the binding of antibody B by at least 60%, particularly at least 70%, and more particularly at least 80% (and vice versa) compared to a positive control lacking one of the antibodies. As one skilled in the art will appreciate, competition can be assessed in a variety of assay settings. One suitable assay involves the use of Biacore technology (e.g., by using a Biacore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interaction using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. Furthermore, a high throughput process for "binning" antibodies based on their cross-competition is described in International Patent Application No. WO2003 / 48731.

[0029] An "inhibitor of expression" refers to a natural or synthetic compound that has the biological effect of inhibiting the expression of a gene.

[0030] The term "endonuclease" refers to an enzyme that breaks phosphodiester bonds in a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively non-specifically (regardless of sequence), while many, typically called restriction endonucleases or restriction enzymes, only cut at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivation generally requires a first step of single- or double-strand breaks in DNA, which can then trigger two distinct cellular mechanisms for DNA repair (error-prone non-homologous end joining (NHEJ) and high-fidelity homology-directed repair (HDR)), which can be exploited to inactivate DNA. The DNA-targeting endonuclease can be a naturally occurring endonuclease (e.g., bacterial meganuclease) or it can be artificially created (e.g., engineered meganucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs), among others).

[0031] In some embodiments, the DNA targeting endonuclease of the present invention is a TALEN. The term "TALEN" as used herein has its general meaning in the art, which refers to a transcription activation-like effector nuclease, an artificial nuclease that can be used to edit a target gene. TALENs are artificially created by fusing a TAL effector ("TALE") DNA binding domain, such as one or more TALEs, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 TALEs, to a DNA modification domain, such as a FokI nuclease domain. Transcription activation-like effectors (TALEs) may be designed to bind to any desired DNA sequence (Zhang (2011), Nature Biotech. 29: 149-153). By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme that is specific for any desired DNA sequence can be created. These can then be introduced into cells where they can be used for genome editing (Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501). TALEs are proteins secreted by Xanthomonas. The DNA binding domain contains a highly conserved 33-34 amino acid sequence that is repeated, except for the 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with the recognition of specific nucleotides. Thus, they can be designed to bind to desired DNA sequences (Zhang (2011), Nature Biotech. 29: 149-153). To generate TALENs, the TALE protein is fused to a nuclease (N), such as a wild-type or mutant FokI endonuclease.Several mutations have been made to FokI in order to use it in TALENs; these improve, for example, cleavage specificity or activity (Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96). The FokI domain functions as a dimer, requiring two constructs with unique DNA domains that bind to sites in the target genome in the proper orientation and spacing. Both the number of amino acid residues between the DNA binding domain of the TALE and the FokI cleavage domain, and the number of bases between the two individual TALEN binding sites, appear to be important parameters for achieving high levels of activity (Miller et al. (2011) Nature Biotech. 29: 143-8). TALENs can be used to generate double-strand breaks in target nucleic acids, for example, at sites within genes, within cells. If the repair mechanism improperly repairs the break via non-homologous end joining, mutations can be introduced at the break site (Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16). For example, improper repair can introduce frameshift mutations. Alternatively, foreign DNA may be introduced into a cell along with the TALEN, depending on the foreign DNA sequence and the chromosomal sequence; this process can be used to modify a target gene via a homologous recombination repair pathway, for example, to correct a defect in the target gene, thereby resulting in expression of the repaired target gene, or, for example, to introduce such a defect into a wild-type gene, thereby reducing expression of the target gene.

[0032] In some embodiments, the DNA targeting endonuclease of the present invention is a ZFN. The term "ZFN" or "zinc finger nuclease" as used herein has its general meaning in the art and refers to a zinc finger nuclease, which is an artificial nuclease that can be used to edit a target gene. Similar to TALENs, ZFNs comprise a DNA modifying domain, such as a nuclease domain, such as a FokI nuclease domain (or a derivative thereof), fused to a DNA binding domain. In the case of ZFNs, the DNA binding domain comprises one or more zinc fingers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160). Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can, for example, contain Cys2His2 and can recognize sequences of about 3 bp. Various zinc fingers with known specificity can be combined to create multi-finger polypeptides that recognize sequences of about 6, 9, 12, 15, or 18 bp. Various selection and modular assembly techniques, including phage display, yeast one-hybrid system, bacterial one-hybrid system and two-hybrid system, and mammalian cells, are available to create zinc fingers (and combinations thereof) that recognize specific sequences. Zinc fingers can be designed to bind to predetermined nucleic acid sequences. Criteria for engineering zinc fingers to bind to predetermined nucleic acid sequences are known in the art (Sera (2002), Biochemistry, 41:7074-7081; Liu (2008) Bioinformatics, 24:1850-1857). ZFNs that use a FokI nuclease domain or other dimeric nuclease domains function as dimers.Therefore, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind to opposite strands of DNA, and their nucleases must be properly spaced apart (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5). Also, like TALEN, ZFNs can cause double-strand breaks in DNA, which, if repaired improperly, can cause frameshift mutations, for example, via non-homologous end joining, thereby reducing the expression of target genes in cells.

[0033] In some embodiments, the DNA targeting endonuclease of the present invention is a CRISPR-associated endonuclease. As used herein, the term "CRISPR-associated endonuclease" has its general meaning in the art and refers to associated clustered regularly interspaced short palindromic repeats, which are segments of prokaryotic DNA that contain short repeated base sequences. In bacteria, CRISPR / Cas loci code for an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). Three types (I-VI) of CRISPR systems have been identified. CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element. CRISPR clusters are transcribed and processed into mature CRISPR (clustered regularly interspaced short palindromic repeats) RNA (crRNA). CRISPR-associated endonucleases Cas9 and Cpf1 belong to type II and type V CRISPR / Cas systems and have strong endonuclease activity to cleave target DNA. Cas9 is guided by mature crRNA (CRISPR RNA) containing a unique target sequence of about 20 nucleotides (called spacer) and a transactivating small RNA (tracrRNA) that serves as a guide for RNase III-assisted processing of the crRNA precursor. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and a complementary sequence on the target DNA (called protospacer). Cas9 recognizes a trinucleotide (NGG) protospacer motif (PAM) to specify the cleavage site (the third or fourth nucleotide from the PAM). The crRNA and tracrRNA can be expressed separately or engineered into an artificially fused small guide RNA (sgRNA) via a synthetic stem-loop to mimic the natural crRNA / tracrRNA duplex.Such sgRNAs, e.g., small hairpin RNAs (shRNAs), can be synthesized or in vitro transcribed for direct RNA transfection or expressed from U6- or H1-driven RNA expression vectors.

[0034] In some embodiments, the CRISPR-associated endonuclease is a Cas9 nuclease. The Cas9 nuclease may have a nucleotide sequence identical to a wild-type Streptococcus pyogenes sequence. In some embodiments, the CRISPR-associated endonuclease may be a sequence from other species, such as other Streptococcus species, such as Thermophilus; Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacterial genomes and archaea, or other prokaryotic microorganisms. Alternatively, the Cas9 sequence of wild-type Streptococcus pyogenes may be modified. The nucleic acid sequence may be codon-optimized for efficient expression in mammalian cells, i.e., "humanized." The humanized Cas9 nuclease sequence may be, for example, a Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank Accession Nos. KM099231.1 GL669193757; KM099232.1 GL669193761; or KM099233.1 GL669193765. Alternatively, the Cas9 nuclease sequence may be, for example, a sequence contained within a commercially available vector, such as pX330, pX260, or pMJ920 from Addgene, Inc. (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease may have an amino acid sequence that is a variant or fragment of any of the Cas9 endonuclease sequences of Genbank Accession Nos. KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765, or the Cas9 amino acid sequence of pX330, pX260, or pMJ920 (Addgene, Cambridge, MA).

[0035] In some embodiments, the CRISPR-associated endonuclease is Cpf1 nuclease. As used herein, the term "Cpf1 protein" refers to Cpf1 wild-type protein from type V CRISPR-Cpf1 system, modified form of Cpf1 protein, mutant of Cpf1 protein, ortholog of Cpf1, and combinations thereof. Cpf1 gene encodes Cpf1 protein with RuvC-like nuclease domain that is homologous to each domain of Cas9, but lacks the HNH nuclease domain present in Cas9 protein.The V-type system includes Parcubacteria bacterium GWC2011_GWC2_44_17 (PbCpf1), Lachnospiraceae bacterium MC2017 (Lb3 Cpf1), Butyrivibrio proteoclasticus (BpCpf1), Peregrinibacteria bacterium GW2011_GWA33_10 (PeCpf1), Acidaminococcus spp. BV3L6 (AsCpf1), Porphyromonas macacae (PmCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), Porphyromonas krevioricanis (PmCpf1), and the Lachnospiraceae bacterium ND2006 (LbCpf1). crevioricanis (PcCpf1), Prevotella disiens (PdCpf1), Moraxella bovoculi 237 (MbCpf1), Smithella spp. SC_K08D17 (SsCpf1), Leptospira inadai (LiCpf1), Lachnospiraceae bacterium MA2020 (Lb2Cpf1), Franciscella novicida U112 (FnCpf1), Candidatus methanoplasma termitum (CMtCpf1), and Eubacterium erythrocytes (ECs) were also detected. eligens) (EeCpf1). Recently, it has been demonstrated that Cpf1 also has ribonuclease activity and is involved in processing of CRISPR RNA (crRNA) precursors (Fonfara, I., et al., "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA," Nature 28; 532(7600):517-21 (2016)).

[0036] The term "treatment" or "treating" as used herein refers to both prophylactic or preventative treatments and curative or disease-modifying treatments, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, including suppression of clinical recurrence. Treatments may be administered to patients who have a medical disorder or who may eventually acquire a disorder, to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurring disorder, or to extend the patient's survival beyond the expected survival in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, such as a dosing pattern used during therapy. Therapeutic regimens can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide the patient with high levels of drug during the initial period of the treatment regimen. An induction regimen may use (partially or entirely) a "loading regimen," which may include administering a higher dose of a drug than the doctor would use during a maintenance regimen, administering a drug more frequently than the doctor would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0037] The term "cancer" as used herein has its general meaning in the art, but is not limited to solid tumors and blood-borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood, and vasculature. The term "cancer" further includes both primary and metastatic cancers. Examples of cancers that can be treated by the methods and compositions of the present invention include, but are not limited to, cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, digestive tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, the cancer may be of the following histological types, among others, but not limited to: malignant tumor; carcinoma; undifferentiated carcinoma; giant cell spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma of familial polyposis coli; solid tumor; malignant carcinoma bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; cutaneous adenocarcinoma organ cancer; apocrine adenocarcinoma; sebaceous adenocarcinoma; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary gland page Whett's disease;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;malignant thymoma;malignant ovarian stromal tumor;malignant theca tumor;malignant granulosa cell tumor;and malignant Sertoli stromal cell tumor;Sertoli cell carcinoma;malignant Leydig cell tumor;malignant lipid cell tumor;malignant paraganglioma;malignant extramammary paraganglioma;pheochromocytoma;angioangiosarcoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma in giant pigmented nevus;epithelioid cell melanoma tumor;malignant blue nevus;sarcoma;fibrosarcoma;malignant fibrous histiocytoma;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;encystic rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian goiter;choriocarcinoma;malignant mesonephroma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;Malignant hemangiopericytoma;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Malignant chondroblastoma;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Malignant odontogenic tumor;Ameloblastic odontosarcoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pinealoma;Chordoma;Malignant glioma;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory nerve tumor;Malignant meningioma;Neurofibroma Fibrosarcoma;malignant neuromyeloma;malignant granular cell tumor;malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;paragranuloma;small lymphocytic lymphoma;diffuse large cell lymphoma;follicular lymphoma;mycosis fungoides;non-Hodgkin's lymphoma not otherwise specified;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia. ;

[0038] The term "tumor tissue sample" as used herein refers to any tissue tumor sample derived from a patient. The tissue sample is obtained for the purpose of in vitro evaluation. In some embodiments, the tumor sample may be obtained from a tumor excised from a patient. In some embodiments, the tumor sample may be obtained from a biopsy performed on the patient's primary tumor or from a biopsy performed on a metastatic tumor distant from the patient's primary tumor. For example, an endoscopic biopsy performed on the intestine of a patient suffering from colorectal cancer. In some embodiments, the tumor tissue sample includes (i) the entire primary tumor (whole), (ii) a tissue sample from the center of the tumor, (iii) a tissue sample from the tissue immediately surrounding the tumor (this tissue may more specifically be named the "invasive margin" of the tumor), (iv) lymph islands in close proximity to the tumor, (v) lymph nodes located closest to the tumor, (vi) a tumor tissue sample collected before surgery (e.g., for patient follow-up after treatment), and (vii) a distant metastasis. "Invasive margin" as used herein has its general meaning in the art and refers to the cellular environment surrounding a tumor. In some embodiments, tumor tissue samples, whether they are from the center of the tumor, from the invasive margin of the tumor, or from the nearest lymph node, include tissue slices or tissue slices taken from the tumor center or from the invasive margin surrounding the tumor, including after surgical tumor removal or after collection of tissue samples for biopsy, for further quantification of one or several biological markers, in particular through histological or immunohistochemical methods, through flow cytometric methods, and through gene or protein expression analysis methods, including genomic and proteomic analysis. Tumor tissue samples can of course be subjected to various well-known post-collection fractionation and preservation techniques (e.g., fixation, preservation, freezing, etc.). Samples can be fresh, frozen, fixed (e.g., formalin-fixed), or embedded (e.g., paraffin-embedded).

[0039] As used herein, the term "enhanced therapeutic efficacy" in relation to cancer refers to a slowing or shrinkage of the growth of cancer cells or solid tumors, a reduction in the total number of cancer cells or total tumor burden. Thus, "improved therapeutic outcome" or "enhanced therapeutic efficacy" means that there is an improvement in the patient's condition according to any clinically accepted criteria, including, for example, reduced tumor size, increased time to tumor progression, increased progression-free survival, increased overall survival, increased life span, or improved quality of life. In particular, "improved" or "enhanced" refers to an improvement or enhancement of 1%, 5%, 10%, 25%, 50%, 75%, 100%, or more than 100% of any clinically accepted indicator of therapeutic outcome or efficacy. As used herein, the term "compared to" when used in the context of comparing the activity and / or efficacy of a combination composition comprising an immune checkpoint inhibitor together with an NRP-1 inhibitor to the activity and / or efficacy of an immune checkpoint inhibitor alone refers to a comparison using amounts known to be equivalent by those skilled in the art.

[0040] As used herein, the term "co-administration" refers to the process of administering a combination of an NRP-1 inhibitor and an immune checkpoint inhibitor to the same patient. The NRP-1 inhibitor and the immune checkpoint inhibitor may be administered simultaneously, substantially simultaneously, or sequentially. When administration is sequential, the NRP-1 inhibitor is administered prior to the immune checkpoint inhibitor. The NRP-1 inhibitor and the immune checkpoint inhibitor do not need to be administered using the same vehicle. The NRP-1 inhibitor and the immune checkpoint inhibitor may be administered one or more times, and the number of administrations of each component of the combination may be the same or different. Furthermore, the NRP-1 inhibitor and the immune checkpoint inhibitor do not need to be administered simultaneously.

[0041] The terms "combination" and "combination therapy" as used are synonymous and refer to a treatment that includes the administration of at least two compounds, administered simultaneously, separately, or sequentially. As used herein, the term "co-administration" as used herein means a process in which a combination of at least two compounds is administered to the same patient. The at least two compounds may be administered simultaneously, substantially simultaneously, or sequentially. The at least two compounds may be administered separately, using different vehicles or compositions. The at least two compounds may also be administered in the same vehicle or composition (e.g., pharmaceutical composition). The at least two compounds may be administered one or more times, and the number of administrations of each component of the combination may be the same or different.

[0042] The term "therapeutically effective amount" as used herein refers to an amount effective at a dosage and for a period of time required to achieve a desired therapeutic result. A therapeutically effective amount of an active agent may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The effective dose and dosage regimen for the active agent will depend on the disease or condition to be treated, and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician can start a dose of the active agent used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. In general, the appropriate dose of the composition of the present invention will be the amount of the compound that is the lowest dose that is effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend on the factors described above. For example, a therapeutically effective amount for use in treatment can be measured by its ability to stabilize the progression of a disease. Typically, the cancer suppression ability of a compound can be evaluated, for example, in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a patient. One of skill in the art would be able to determine such amounts based on such factors as the size of the patient, the severity of the patient's symptoms, and the particular composition or route of administration selected. An exemplary non-limiting range for a therapeutically effective amount of an inhibitor of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, such as about 0.5, such as about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. An exemplary non-limiting range for a therapeutically effective amount of an inhibitor of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg, or 0.1-3 mg / kg, such as about 0.5-2 mg / kg.Administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, e.g., administered near the target site. The dosage regimen in the above treatment and use methods is adjusted to give the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as dictated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of the treatment is monitored during the treatment, e.g., at predetermined time points. In some embodiments, the effectiveness may be monitored by visualization of the diseased area or by other diagnostic methods further described herein, e.g., by performing one or more PET-CT (positron emission tomography-computed tomography) scans using labeled inhibitors, fragments, or mini-antibodies derived from the inhibitors of the invention. If desired, an effective daily amount of the pharmaceutical composition may be administered as two, three, four, five, six, or more divided doses at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the human monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time, for example, over 24 hours or more, to minimize any undesirable side effects. Effective doses of the inhibitors of the invention may also be administered using a dosing period of once a week, once every two weeks, or once every three weeks. The dosing period may be limited, for example, to 8 weeks, 12 weeks, or until clinical progress is established.As a non-limiting example, treatment according to the present invention may be performed in an amount of about 0.1 to 100 mg / kg per day of an inhibitor of the present invention, e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 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, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg per day. The dose may be provided using a single or divided dose every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, on at least one of the 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, for at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks after initiation of treatment, or any combination thereof.

[0043] The term "cancer vaccine" as used herein has its general meaning in the art and refers to a composition that can induce active immunity against at least one cancer antigen. Cancer vaccines can produce antibodies or simply activate certain cells, particularly antigen-presenting cells, T lymphocytes (especially CD8 positive T cells) and B lymphocytes. Cancer vaccines can be compositions for prophylactic or therapeutic purposes or both.

[0044] The term "antigen" as used herein refers to a molecule that can specifically bind to an antibody or to a T cell receptor (TCR) when processed and presented by an MHC molecule. The term "antigen" as used herein also encompasses T cell epitopes. An antigen can further be recognized by the immune system and / or can induce a humoral and / or cellular immune response, which activates B and / or T lymphocytes. An antigen may have one or more epitopes or antigenic sites (B and T epitopes). The term "cancer antigen" as used herein refers to an antigen that is characteristic of tumor tissue. Examples of cancer antigens include carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), HER-2 / neu, BAGE, GAGE, MAGE1-4, 6 and 12, MUC-related proteins (mucins) (MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), MARCO-MART, cyclin B1, cyclin D, Pmel 17 (gpl00), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), prostate Capsm, prostate serum antigen (PSA), PRAME (melanoma antigen), β-catenin, MUM-1-B (a ubiquitously mutated gene product in melanoma), GAGE ​​(melanoma antigen) 1, BAGE (melanoma antigen) 2-10, C-ERB2 (Her2 / neu), EBNA (Epstein-Barr virus nuclear antigen) 1-6, gp75, human papillomavirus (HPV) E6 and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67.In some embodiments, the antigen is selected from tumor associated antigens, including antigens derived from leukemias and lymphomas, neurological tumors such as astrocytoma or glioblastoma, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors, stomach cancer, colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such as cervical cancer, uterine cancer, ovarian cancer, vaginal cancer, testicular cancer, prostate cancer, or penile cancer, bone tumors, vascular tumors, or cancer of the lip, nasopharyngeal cancer, pharynx and oral cavity cancer, esophageal cancer, rectal cancer, bladder cancer, biliary system cancer, laryngeal cancer, lung and bronchial cancer, bladder cancer, kidney cancer, brain cancer, and cancers of other parts of the nervous system, thyroid cancer, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, and leukemia.

[0045] As used herein, the term "immunoadjuvant" refers to a compound that can induce and / or enhance an immune response to an antigen when administered to a subject or animal. It is also intended to mean a substance that generally acts to accelerate, prolong, or enhance the quality of a particular immune response to a particular antigen.

[0046] As used herein, the term "responder" in the context of this disclosure refers to a patient who will achieve a response, i.e., a patient whose cancer is cured, regressed, or ameliorated. According to the present invention, the term "non-responder" also includes patients with stabilized cancer.

[0047] Methods to increase the amount of tumor-infiltrating CD8+ T cells: A first object of the present invention relates to a method for increasing the amount of tumor-infiltrating CD8-positive T cells in a patient suffering from cancer, comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor.

[0048] In some embodiments, the NRP-1 inhibitor is an antibody having binding affinity for NRP-1.

[0049] In some embodiments, the NRP-1 inhibitor is an antibody against the extracellular domain of NRP-1.

[0050] In some embodiments, the antibody allows cytotoxic T cells to internalize NRRP-1.

[0051] In some embodiments, the antibody binds to domain c of NRP-1. In some embodiments, the antibody of the invention is capable of inhibiting binding of NRP-1 to semaphorin 3A.

[0052] In some embodiments, the NRP-1 inhibitor is an antibody that has binding affinity for a region of NRP-1 that binds to semaphorin 3A.

[0053] In some embodiments, the NRP-1 inhibitor is an antibody having binding affinity for an amino acid sequence ranging from amino acid residue 1 to amino acid residue 280 of SEQ ID NO:1.

[0054] In some embodiments, the antibody does not inhibit binding of VEGF to NRP-1.

[0055] In some embodiments, the NRP-1 inhibitor is an antibody that has binding affinity for semaphorin 3A.

[0056] In some embodiments, the NRP-1 inhibitor is an antibody that has binding affinity for the domain of semaphorin 3A that binds to NRP-1.

[0057] In some embodiments, the antibody is a humanized antibody. Humanization methods include, but are not limited to, those described in U.S. Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference.

[0058] In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies can also be prepared by immunizing mice genetically modified for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

[0059] In some embodiments, the antibodies of the invention are single chain antibodies.

[0060] In some embodiments, the antibody is derived from the anti-NRP1 antibody YW64.3 described in Mol. Biol. (2007) 366, 815-829 and in U.S. Patent Application No. 8378080B1. In particular, the anti-NRP-1 antibody according to the invention is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); Includes.

[0061] In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9. In some embodiments, the anti-NRP-1 antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 10. In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9 and a heavy chain variable domain sequence of SEQ ID NO: 10.

[0062] [ka]

[0063] In some embodiments, the anti-NRP-1 antibodies of the invention a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); for binding to the NRP-1 isoform.

[0064] In some embodiments, the antibody comprises a human heavy chain constant region sequence but will not induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the antibody of the invention does not comprise an Fc domain capable of substantially binding to an FcgRIIIA (CD16) polypeptide. In some embodiments, the antibody of the invention lacks an Fc domain (e.g., lacks a CH2 domain and / or a CH3 domain) or comprises an Fc domain of an IgG2 or IgG4 isotype. In some embodiments, the antibody of the invention consists of or comprises a Fab, Fab', Fab'-SH, F(ab')2, Fv, diabody, single chain antibody fragment, or a multispecific antibody comprising multiple different antibody fragments. In some embodiments, the antibody of the invention is not linked to a toxic moiety. In some embodiments, one or more selected amino acids from the amino acid residues may be replaced with a different amino acid residue such that the antibody exhibits altered binding to C2q and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in US Pat. No. 6,194,551 by dusogie et al.

[0065] In some embodiments, the NRP-1 inhibitors are polypeptides that contain the functional equivalents of NRFP-1, respectively. For example, functional equivalents include molecules that bind to semaphorin 3A and contain all or part of the extracellular domain of NRP-1, thereby forming a soluble receptor that can capture semaphorin 3A. Typically, functional equivalents are at least 80% homologous to the corresponding protein.

[0066] In some embodiments, a functional equivalent is at least 90% homologous as assessed by any conventional analytical algorithm. Thus, the present invention provides a polypeptide capable of inhibiting binding of NRP-1 to semaphorin 3A, the polypeptide comprising consecutive amino acids having a sequence corresponding to the sequence of at least a portion of the extracellular domain of NRP-1, which portion binds to semaphorin 3A.

[0067] In some embodiments, the polypeptide comprises the extracellular domain of NRP-1. In some embodiments, the polypeptide comprises an amino acid sequence comprising domain c of NRP-1.

[0068] In some embodiments, the polypeptide comprises an amino acid sequence comprising the transmembrane domain of NRP-1.

[0069] In some embodiments, the polypeptide comprises the amino acid sequence ranging from amino acid residue 1 to amino acid residue 280 of SEQ ID NO:1.

[0070] In some embodiments, the polypeptide does not include a moiety that binds to VEGF.

[0071] In some embodiments, the polypeptide comprises a functional equivalent of NRP-1, which is fused to an immunoglobulin constant domain (Fc region) to form an immunoadhesin. Immunoadhesins can have many of the valuable chemical and biological properties of human antibodies. Immunoadhesins can be constructed from human protein sequences with the desired specificity linked to appropriate human immunoglobulin hinge and constant domain (Fc) sequences, so that the desired binding specificity can be achieved using fully human components. The immunoglobulin sequence is typically, but not necessarily, an immunoglobulin constant domain. The immunoglobulin portion in the chimeras of the invention can be derived from IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM, but typically from IgG1 or IgG3. In some embodiments, the functional equivalent of PD-1 or NRP-1 and the immunoglobulin sequence portion of the immunoadhesin are linked by a minimal linker.

[0072] In some embodiments, the NRP-1 inhibitor is an NRP-1 expression inhibitor.

[0073] In some embodiments, the gene expression inhibitor is a small interfering RNA (siRNA), an antisense oligonucleotide, or a ribozyme. For example, antisense oligonucleotides (including antisense RNA molecules and antisense DNA molecules) may act to directly block the translation of NRP-1 mRNA by binding to NRP-1 mRNA, thereby preventing protein translation or increasing the degradation of mRNA, thereby reducing the level of NRP-1 in cells, thereby reducing activity. For example, antisense oligonucleotides that are at least about 15 bases long and complementary to unique regions of the mRNA transcript sequence encoding NRP-1 can be synthesized, for example, by conventional phosphodiester technology. Methods for using antisense technology to specifically inhibit gene expression of genes whose sequences are known are well known in the art (see, for example, U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as expression inhibitors for use in the present invention. Gene expression of NRP-1 can be reduced by contacting a patient or cells with small double-stranded RNA (dsRNA) or a vector or construct that causes the production of small double-stranded RNA, whereby gene expression of NRP-1 is specifically inhibited (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs, and ribozymes of the present invention can be delivered in vivo alone or in combination with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the introduction of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid into a cell, typically a cell expressing NRP-1. Typically, the vector transports the nucleic acid into the cell with reduced degradation compared to the extent of degradation that would occur in the absence of the vector.In general, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that are engineered by inserting or incorporating antisense oligonucleotides, siRNA, shRNA, or ribozyme nucleic acid sequences.Viral vectors are a preferred type of vector, including, but not limited to, nucleic acid sequences derived from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40-type viruses; polyoma viruses; Epstein-Barr virus; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses, such as retroviruses.Other vectors that are not named but are known in the art can also be easily used.

[0074] In some embodiments, the expression inhibitor is an endonuclease.

[0075] In a particular embodiment, the endonuclease is CRISPR-cas.

[0076] In some embodiments, the endonuclease is CRISPR-cas9 from Streptococcus pyogenes. CRISPR / cas9 systems are described in U.S. Patent Application No. 8697359B1 and U.S. Patent Application No. 2014 / 0068797. In some embodiments, the endonuclease is CRISPR-Cpf1, which is CRISPR1 (Cpf1) from Prevotella and Francisella, most recently characterized in Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0077] 1. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor: A further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a pharma- ceutical effective amount of an NRP-1 inhibitor.

[0078] In some embodiments, the NRP-1 inhibitor is an anti-NRP-1 antibody derived from the anti-NRP1 antibody YW64.3 described in Mol. Biol. (2007) 366, 815-829 and U.S. Patent Application No. 8378080B1. In particular, the anti-NRP-1 antibody is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); Includes.

[0079] In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9. In some embodiments, the anti-NRP-1 antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 10. In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9 and a heavy chain variable domain sequence of SEQ ID NO: 10.

[0080] In some embodiments, the NRP-1 inhibitor is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); and an anti-NRP-1 antibody that cross-competes for binding to an NRP-1 isoform with an antibody comprising:

[0081] In particular, the method of the present invention is particularly suitable for treating the cancer characterized by low tumor infiltration of CD8 positive T cells.Typically, the tumor infiltration of CD8 positive T cells is determined by any conventional method in the art.For example, the determination comprises quantifying the density of CD8 positive T cells in tumor samples obtained from patients.

[0082] In some embodiments, the quantification of the density of CD8 positive T cells is determined by immunohistochemistry (IHC). For example, the quantification of the density of CD8 positive T cells is performed by contacting the tumor tissue sample with a binding pair (e.g., an antibody) specific for a cell surface marker of the cells. Typically, the quantification of the density of CD8 positive T cells is performed by contacting the tumor tissue sample with a binding pair (e.g., an antibody) specific for CD8. Typically, the density of CD8 positive T cells is measured as the number of these cells counted per unit of surface area of ​​the tissue sample, e.g., per cm of surface area of ​​the tumor tissue sample. 2 or 1mm 2 In some embodiments, cell density is also expressed as the number of cells counted per unit of sample volume, e.g., per cm of a tumor tissue sample. 3Cell density may be expressed as the number of cells per 100 cells. In some embodiments, cell density may also consist of the ratio of a particular cell per total cells (set at 100%). Immunohistochemistry typically includes the following steps: i) fixation of tumor tissue samples with formalin, ii) embedding of the tumor tissue samples in paraffin, iii) cutting of the tumor tissue samples into sections for staining, iv) incubating the sections with a binding pair specific for the marker, v) rinsing the sections, vi) incubating the sections with a secondary antibody, typically biotinylated, and vii) revealing the antigen-antibody complex, typically using an avidin-biotin-peroxidase complex. Thus, the tumor tissue sample is first incubated with the binding pair. After washing, the labeled antibody bound to the marker of interest is revealed by an appropriate technique, depending on the type of label (e.g., radioactive, fluorescent, or enzymatic) generated by the labeled antibody. Alternatively, the method of the present invention may use a secondary antibody conjugated to an amplification system (to enhance the staining signal) and an enzyme molecule. Such conjugated secondary antibodies are commercially available, for example, from Dako, EnVision system. Counterstains, such as hematoxylin and eosin, DAPI, Hoechst, etc., may be used. Other staining methods may be accomplished using any suitable method or system, including automated, semi-automated, or manual systems, as would be apparent to one of skill in the art. For example, one or more labels may be attached to the antibody, allowing for detection of the target protein (i.e., marker). Exemplary labels include radioisotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to the primary and / or secondary affinity ligands include fluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescent compounds (e.g., luminal, imidazole) and bioluminescent proteins (e.g., luciferin, luciferase), haptens (e.g., biotin).A wide variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke JR (1972) Annu. Rev. Biochem. 41:843-868. Affinity ligands may also be used, including enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-lactamase), radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S, or 125I) and particles (e.g., gold). Various types of labels can be conjugated to the affinity ligand using various chemical reactions, such as amine or thiol reactions. However, other reactive groups other than amines and thiols, such as aldehydes, carboxylic acids, and glutamines, can also be used. Various enzymatic staining methods are known in the art for detecting proteins of interest. For example, enzymatic interactions can be visualized using peroxidase, alkaline phosphatase, or different chromogens, such as diaminobenzidine (DAB), aminoethylcarbazole (AEC), or fast red. In other examples, antibodies can be conjugated to peptides or proteins, which can be detected via labeled binding pairs or antibodies. In indirect immunohistochemical assays, a secondary antibody or secondary binding pair is required to detect the binding of the first binding pair, since it is not labeled. The resulting stained specimens are each imaged using a system for viewing detectable signals and acquiring images, such as stained digital images. Methods for acquiring images are well known to those skilled in the art. For example, once a sample is stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as an upright or inverted optical microscope, a scanning confocal microscope, a camera, a scanning or tunneling electron microscope, a scanning probe microscope, and an infrared image detector. In some instances, the image can be acquired digitally. The resulting image can then be used to quantitatively or semi-quantitatively determine the amount of marker in the sample. A variety of automated sample processing, scanning, and analysis systems suitable for use in immunohistochemistry are available in the art. Such systems may include automated staining and microscope scanning, computerized image analysis, comparison of serial sections (to control for variations in sample orientation and size), generation of digital reports, and archiving and tracking of samples (such as slides on which tissue sections are placed).Cell imaging systems are commercially available that combine conventional optical microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, for example, the CAS-200 system (Becton Dickinson). In particular, detection may be performed manually or by image processing techniques involving computer processors and software. For example, using such software, images may be constructed, calibrated, normalized, and / or verified based on factors including, for example, staining quality or staining intensity, using procedures known to those skilled in the art (see, for example, published U.S. Patent Publication No. 20100136549). Images may be quantitatively or semi-quantitatively analyzed and may be scored based on the staining intensity of the sample. Quantitative or semi-quantitative histochemistry refers to a method in which a histochemistry-subjected sample is scanned and scored to identify and quantify the presence of a particular biomarker (i.e., marker). Quantitative or semi-quantitative methods may use imaging software to detect staining density or amount of staining, or methods that detect staining by the naked human eye, where a trained operator ranks the results numerically. For example, images can be quantitatively analyzed using pixel counting algorithms (e.g., Aperio Spectral software, automated quantitative analysis platforms (AQUA® platform), and other standard methods to measure or quantify or semi-quantify the intensity of staining; see, e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S. Patent Publication Nos. 20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328. The ratio of strongly positive staining (e.g., brown staining) to the sum of all stained areas can be calculated and scored. The amount of biomarker (i.e., marker) detected is quantified and presented as a percentage of positive pixels and / or score.For example, the amount may be quantified as a percentage of positive pixels. In some examples, the amount is quantified as a percentage of the stained area, e.g., a percentage of positive pixels. For example, a sample may have at least, or approximately at least, or approximately 0, 1%, 2%, 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%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels compared to the total stained area. In some embodiments, the sample is given a score, which is a numerical indication of the intensity or amount of histochemical staining of the sample and indicates the amount of target biomarker (e.g., marker) present in the sample. The numerical value of absorbance or area percentage can be given a scaled score, for example, on an integer scale. Thus, in some embodiments, the method of the present invention comprises the steps of: i) preparing one or more immunostained tissue section slices obtained by an automated slide staining system by using a binding pair (e.g., an antibody as described above) that can selectively interact with the marker; ii) proceeding with the digitization of the slide of step a. by high-resolution scan capture; iii) detecting the tissue section slices on the digital photograph; iv) preparing a size reference grid with uniformly distributed units having the same surface area (the grid is adapted to the size of the tissue section to be analyzed); and v) detecting, quantifying, and measuring the intensity of stained cells within each unit (thereby evaluating the number or density of stained cells in each unit).

[0083] In some embodiments, the cell density of CD8 positive T cells in the entire tumor tissue sample is determined, the cell density of CD8 positive T cells in the invasive margin or center of the tumor tissue sample is determined, or the cell density of CD8 positive T cells in both the center and invasive margin of the tumor tissue sample is determined.

[0084] Therefore, a further object of the present invention relates to a method for treating cancer in a patient in need thereof, comprising: i) quantifying the density of CD8 positive T cells in a tumor tissue sample obtained from the patient; ii) comparing the density quantified in step i) with a predefined reference value; and iii) administering a therapeutically effective amount of an NRP-1 inhibitor to the patient.

[0085] Typically, the predetermined reference value correlates with the survival time of the patient. Those skilled in the art will recognize that overall survival is generally based on and expressed as the proportion of people who survive a particular type of cancer for a particular period of time. Cancer statistics often use the 5-year overall survival rate. Generally, overall survival rate does not specify whether cancer survivors are still undergoing treatment or are cancer-free (achieved remission) at 5 years. Disease-free survival rate (DFS) gives more specific information and is the number of people with a particular cancer who have achieved remission. Also, progression-free survival (PFS) rate (the number of people who still have cancer but their disease has not progressed) includes people who may have had some success with treatment but the cancer has not disappeared completely. As used herein, the expression "short survival time" indicates that the patient will have a survival time that will be shorter than the median (or mean) observed in the general population of patients suffering from said cancer. If the patient will have a short survival time, it means that the patient will have a "bad prognosis". Conversely, the phrase "long survival" indicates that a patient will have a survival that will be longer than the median (or mean) observed in the general population of patients suffering from said cancer. If a patient will have a long survival, it means that the patient will have a "good prognosis."

[0086] In some embodiments, the predetermined numerical value is a threshold or cut-off value. Typically, the "threshold" or "cut-off value" can be experimentally, empirically, or theoretically determined. The threshold value may also be freely selected based on existing experimental and / or clinical conditions, as would be recognized by a person skilled in the art. For example, retrospective measurements of cell density of appropriately deposited historical patient samples may be used to establish the predetermined reference value. The threshold value must be determined so that optimal sensitivity and specificity are obtained according to the function of the test and the benefit / risk balance (false positive and false negative clinical outcomes). Typically, optimal sensitivity and specificity (and also threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the density of CD8 positive T cells in a reference group, an algorithmic analysis is used for the statistical treatment of the density measured in the sample to be tested, which allows to obtain a classification standard with significance for the classification of the sample. The full name of the ROC curve is the receiver operator characteristic curve, which is also known as the receiver operation characteristic curve. It is mainly used for biochemical clinical diagnostic tests. The ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). The ROC curve uses image synthesis to reveal the relationship between sensitivity and specificity. A series of various cutoff values ​​(thresholds or critical values, i.e., boundary values ​​between normal and abnormal results of a diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then, to draw the curve, sensitivity is used as the vertical axis coordinate and specificity is used as the horizontal axis coordinate. The larger the area under the curve (AUC), the higher the accuracy of the diagnosis. On the ROC curve, the point closest to the left end of the coordinate diagram is the critical point, which has both high sensitivity and high specificity values. The AUC value of the ROC curve is 1.0 to 0.5. If AUC>0.5, the diagnostic result is better as the AUC approaches 1.If the AUC is between 0.5 and 0.7, the accuracy is low. If the AUC is between 0.7 and 0.9, the accuracy is moderate. If the AUC is greater than 0.9, the accuracy is very high. The algorithmic method is preferably implemented using a computer. Existing software or systems in the art can be used to draw the ROC curve, such as MedCalc9.2.0.1 medical statistics software, SPSS9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.AS, GB STAT VI0.0 (Dynamic Microsystems, Silver Spring, MD, USA), etc.

[0087] In some embodiments, the predetermined reference value comprises the steps of: a) preparing a collection of tumor tissue samples from patients suffering from a cancer of interest; b) for each tumor tissue sample prepared in step a) providing information on the actual clinical outcome for the corresponding patient (i.e., disease-free survival (DFS) and / or overall survival (OS)); c) providing a series of arbitrary quantitative values; d) quantifying the density of CD8 positive T cells for each tumor tissue sample contained in the collection prepared in step a); e) classifying the tumor tissue samples into two groups, respectively, at one specific arbitrary quantitative value provided in step c); (i) the first group comprises tumor tissue samples exhibiting a lower level of a quantitative value than said arbitrary quantitative value comprised in said series of quantitative values; (ii) the second group comprises tumor tissue samples exhibiting a lower level of a quantitative value than said arbitrary quantitative value comprised in said series of quantitative values; , tumor tissue samples that exhibit a higher level of a quantitative value than any of the quantitative values ​​included in the series of quantitative values; whereby, for said specific quantitative value, two groups of tumor tissue samples are obtained, where the tumor tissue samples of each group are counted separately; f) calculating the statistical significance between (i) the quantitative value obtained in step e) and (ii) the actual clinical outcome of the patient from which the tumor tissue samples included in the first group and the second group provided in step f) originate; g) repeating steps f) and g) until any of the quantitative values ​​provided in step d) have been tested; h) setting said predetermined reference value as consisting of any of the quantitative values ​​(the highest statistical significance (most significant) calculated in step g). For example, the density of CD8 positive T cells is evaluated for 100 tumor tissue samples of 100 patients. The 100 samples are ranked according to the density of CD8 positive T cells. Sample 1 has the highest density and sample 100 has the lowest density. The first grouping gives two subsets: sample number 1 on one side and 99 other samples on the other side. The next grouping gives samples 1 and 2 on one side and the 98 remaining samples on the other side, and so on until the final grouping: samples 1-99 on one side and sample number 100 on the other side. Kaplan-Meier curves are constructed for each of the 99 groups of the two subsets according to the information on the actual clinical outcomes for the corresponding cancer patients.Also, the p-value between both subsets was calculated for each of the 99 groups. The predetermined reference value is selected so that the discrimination based on the minimum p-value criterion is strongest. In other words, the density of CD8 positive T cells corresponding to the boundary between both subsets where the p-value is the smallest is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median cell density. Thus, in some embodiments, the predetermined reference value thus allows the discrimination between a bad prognosis and a good prognosis in terms of disease-free survival and overall survival for the patient. In fact, a high statistical significance value (e.g. a low P-value) is generally obtained not only for one arbitrary quantitative value, but also for a series of consecutive arbitrary quantitative values. Therefore, in one alternative embodiment of the present invention, instead of using a fixed predetermined reference value, a series of values ​​is provided. Thus, a minimum statistically significant value (minimum significant threshold, e.g. maximum P-value threshold) is arbitrarily set, and a series of multiple arbitrary quantitative values ​​where the statistically significant value calculated in step g) is higher (more significant, e.g. lower P-value) is retained, thus providing a series of quantitative values. This range of quantitative values ​​includes the "cut-off" value as described above. For example, according to this particular embodiment of the "cut-off" value, the outcome can be determined by comparing the density of CD8 positive T cells with the range of values ​​identified. In some embodiments, the cut-off value thus consists of a range of quantitative values, for example centered on the quantitative value where the highest statistically significant value is found (for example, where the smallest p-value is generally found).

[0088] A method for enhancing the efficacy of an immune checkpoint inhibitor administered to a patient as part of a treatment regimen: A further object of the present invention relates to a method for enhancing the efficacy of an immune checkpoint inhibitor administered to a patient as part of a treatment regimen, said method comprising administering to the patient a pharma- ceutical effective amount of an NRP-1 inhibitor in combination with the immune checkpoint inhibitor.

[0089] In some embodiments, the NRP-1 inhibitor is an anti-NRP-1 antibody derived from the anti-NRP1 antibody YW64.3 described in Mol. Biol. (2007) 366, 815-829 and U.S. Patent Application No. 8378080B1. In particular, the anti-NRP-1 antibody is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); Includes.

[0090] In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9. In some embodiments, the anti-NRP-1 antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 10. In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9 and a heavy chain variable domain sequence of SEQ ID NO: 10.

[0091] In some embodiments, the NRP-1 inhibitor is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); and an anti-NRP-1 antibody that cross-competes for binding to an NRP-1 isoform with an antibody comprising:

[0092] In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-BTLA antibody, and an anti-B7H6 antibody.

[0093] Examples of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; ​​6,682,736; 6,984,720; and 7,605,238. One anti-CTLA-4 antibody is tremelimumab (ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (10D1, also known as MDX-D010), which is a fully human monoclonal IgG antibody that binds to CTLA-4.

[0094] Other immune checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble immunoglobulin fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune checkpoint inhibitors include B7 inhibitors, such as B7-H3 inhibitors and B7-H4 inhibitors. In particular, MGA271, an anti-B7-H3 antibody (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM (T cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). The term "TIM-3" as used herein has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is Galectin 9 (Gal9). Thus, the term "TIM-3 inhibitor" as used herein refers to a compound, substance, or composition capable of inhibiting the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, can modulate or block the signaling pathway of TIM-3, and / or can block the binding of TIM-3 to Galectin-9. Antibodies with specificity for TIM-3 are known in the art and are typically the antibodies described in WO2011155607, WO2013006490, and WO2010117057.

[0095] In some embodiments, the immune checkpoint inhibitor is an IDO (indoleamine-2,3-dioxygenase) inhibitor. Examples of IDO inhibitors are described in WO2014150677. Examples of IDO inhibitors include, but are not limited to, 1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine, β-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyltryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-bromo-4-chloro-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3-amino-naphthoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole, brassinin derivatives, thiohydantoin derivatives, β-carboline derivatives, or brassilexin derivatives. Preferably, the IDO inhibitor is selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphthoic acid, and β-[3-benzo(b)thienyl]-alanine, or a derivative or prodrug thereof.

[0096] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. Thus, as used herein, the term "PD-1 inhibitor" refers to a compound, substance, or composition that can inhibit the function of PD-1. For example, the inhibitor can inhibit the expression or activity of PD-1, modulate or block the signal transduction pathway of PD-1, and / or block the binding of PD-1 to PD-L1 or PD-L2.

[0097] In some embodiments, the PD-1 inhibitor is an antibody directed against the extracellular domain of PD-1. In some embodiments, the PD-1 inhibitor is an antibody directed against the extracellular domain of PD-L1. Examples of PD-1 and PD-L1 antibodies are described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT published patent application numbers: WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699. In some embodiments, PD-1 blocking agents include anti-PD-L1 antibodies. In some embodiments, PD-1 blockers include anti-PD-1 antibodies and similar binding proteins, such as nivolumab (MDX1106, BMS936558, ONO4538), a fully human IgG4 antibody that binds to PD-1 and blocks its activation by its ligands PD-L1 and PD-L2; pembrolizumab (MK-3475 or SCH900475), a humanized monoclonal IgG4 antibody against PD-1; CT-011 humanized antibody that binds to PD-1; AMP-224 is a fusion protein of B7-DC; antibody Fc portions; and BMS-936559 (MDX-1105-01) for blockade of PD-L1 (B7-H1). In some embodiments, the anti-PD-1 antibody is the anti-PD1 antibody Gepi135c as disclosed in WO 2016020856 and Fenwick, Craig, et al. "Tumor suppression of novel anti-PD-1 antibodies mediated through CD28 costimulatory pathway." Journal of Experimental Medicine (2019): jem-20182359.

[0098] In some embodiments, the anti-PD-1 antibody comprises the VH and VL domains of pembrolizumab. In some embodiments, the anti-PD-1 antibody comprises the VH domain of SEQ ID NO: 11 and the VL domain of SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO: 13 and / or a light chain of SEQ ID NO: 14.

[0099] [ka]

[0100] In some embodiments, the anti-PD-1 antibody comprises the VH and VL domains of nivolumab. In some embodiments, the anti-PD-1 antibody comprises the VH domain of SEQ ID NO: 15 and the VL domain of SEQ ID NO: 16. In some embodiments, the anti-PD-1 antibody comprises a heavy chain of SEQ ID NO: 17 and / or a light chain of SEQ ID NO: 18.

[0101] [ka]

[0102] In some embodiments, the PD-1 inhibitor is a small molecule or a peptide or peptide derivative, such as those described in U.S. Patent Nos. 8,907,053; 9,096,642; and 9,044,442, and U.S. Patent Application Publication No. 2015 / 0087581; 1,2,4 oxadiazole compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015 / 0073024; cyclic peptidomimetic compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015 / 0073042; cyclic compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2015 / 0125491; 1,3,4 oxadiazole and 1,3,4 thiadiazole compounds and derivatives, such as those described in International Patent Application Publication No. WO 2015 / 033301; peptide-based compounds and derivatives, such as those described in International Patent Application Publication Nos. WO 2015 / 036927 and WO 2015 / 04490; or macrocyclic peptide-based compounds and derivatives, such as those described in U.S. Patent Application Publication No. 2014 / 0294898; the disclosures of each of which are incorporated herein by reference in their entireties.

[0103] A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective combination of an NRP-1 inhibitor and an immune checkpoint inhibitor: A further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective combination of an NRP-1 inhibitor and an immune checkpoint inhibitor, wherein administration of the combination results in enhanced therapeutic efficacy compared to administration of the immune checkpoint inhibitor alone.

[0104] A multispecific antibody comprising at least one binding site that specifically binds to an immune checkpoint molecule and at least one binding site that specifically binds to NRP-1: A further object of the present invention relates to a multispecific antibody comprising at least one binding site that specifically binds to an immune checkpoint molecule and at least one binding site that specifically binds to NRP-1.

[0105] Multispecific antibodies are typically described in WO2011159877. According to the present invention, the multispecific antibodies of the present invention bind to the extracellular domain of an immune checkpoint molecule (e.g., PD-1) and the extracellular domain of NRP-1. Exemplary formats for the multispecific antibody molecules of the present invention include: (i) two antibodies crosslinked by chemical heteroconjugation, one with specificity for PD-1 and the other with specificity for NRP-1; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single-chain antibody comprising two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain contains two variable domains in tandem through a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010);(v) chemically linked bispecific (Fab')2 fragments;(vi) Tandabs, where the fusion of two single-chain diabodies results in tetravalent bispecific antibodies with two binding sites for each target antigen;(vii) flexibodies, where the combination of scFvs and diabodies results in multivalent molecules;(viii) so-called "dock and lock" molecules based on the "dimerization and docking domain" of protein kinase A, which when applied to Fabs can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to one different Fab fragment;(ix) so-called scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (x) diabodies. Another exemplary type of bispecific antibody is an IgG-like molecule with complementary CH3 domains that force heterodimerization. Such molecules can be prepared using known techniques, such as Triomab / Quadroma (Trion Pharma / Fresenius Biotec), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), strand-exchange engineered domain bodies (SEED bodies) (EMD Serono), Biclonic (Mers), and Duobody (Genmab) technologies. In some embodiments, bispecific antibodies are obtained or can be obtained via controlled Fab arm exchange, typically using duobody technology. Methods for the in vitro generation of bispecific antibodies by controlled Fab arm exchange are described in WO2008119353 and WO2011131746 (both by Genmab Inc.).In one exemplary method described in WO2008119353, bispecific antibodies are formed by exchange of "Fab arms" or "molecular halves" (switching of light chains attached with heavy chains) between two monospecific antibodies, both of which contain an IgG4-like CH3 region, upon incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences. In another exemplary method described in WO2011131746, a bispecific antibody of the invention is prepared by a method comprising the steps of: a) providing a first antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, the Fc region comprising a second CH3 region; wherein the sequences of the first and second CH3 regions are different and a heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than a homodimeric interaction of each of the first and second CH3 regions; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining the bispecific antibody, wherein the first antibody is an antibody of the invention and the second antibody has a different binding specificity, or vice versa. Reducing conditions can be provided by adding a reducing agent, e.g., selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. Step d) can further include restoring the conditions to non-reducing or less reducing, e.g., by removing the reducing agent, e.g., by desalting. Preferably, the sequences of the first and second CH3 regions are different and contain only a few fairly conservative asymmetric mutations, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction of each of the first and second CH3 regions. More details regarding these interactions and how they can be achieved are provided in WO2011131746, which is incorporated herein by reference in its entirety.In some other embodiments, the bispecific antibody of the invention is a symmetric bispecific antibody of class IgG4, comprising two heavy chains, each comprising a variable domain, a CH1 domain and a hinge region, where in each heavy chain: a cysteine ​​in the CH1 domain forming an interchain disulfide bond with a cysteine ​​in the light chain is replaced with another amino acid; optionally, one or more of the amino acids located at the top of the hinge region are replaced with cysteines, where the sequences of the constant regions of each heavy chain are similar or identical, and the variable regions in each heavy chain are different. Such bispecific type antibodies are described in International Patent Application WO2013124450. In some embodiments, the bispecific antibody of the invention is an asymmetric antibody comprising two heavy chains or heavy chain fragments, each comprising at least one variable region, one hinge region and one CH1 domain, wherein the first heavy chain or fragment thereof is of class IgG4 and wherein a) the interchain cysteine ​​at position 127, numbered according to the Kabat numbering system, in the CH1 domain is replaced by another amino acid; and b) optionally one or more of the amino acids located in the upper hinge region are replaced by cysteines, wherein the second heavy chain or fragment thereof is characterized in that part or all of the chain has an amino acid sequence different from that of the first heavy chain in at least a region outside the variable region (e.g. the constant region). Such bispecific antibodies are described in International Patent Application WO2013124451.

[0106] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the invention comprises a first binding site that specifically binds NRP-1 and comprises a light chain variable domain comprising the following amino acid sequences of the complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8).

[0107] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the invention comprises a first binding site that specifically binds to NRP-1 and comprises a light chain variable domain (VL) sequence of SEQ ID NO:9 and a heavy chain variable domain (VH) sequence of SEQ ID NO:10.

[0108] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the invention comprises a second binding site that specifically binds to PD-1 and comprises a VH domain of SEQ ID NO:11 and a VL domain of SEQ ID NO:12.

[0109] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the invention comprises a second binding site that specifically binds to PD-1 and comprises a VH domain of SEQ ID NO:15 and a VL domain of SEQ ID NO:16.

[0110] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the invention comprises: - a first binding site which specifically binds to NRP-1 and comprises a light chain variable domain (VL) sequence of SEQ ID NO: 9 and a heavy chain variable domain (VH) sequence of SEQ ID NO: 10; and - a second binding site which specifically binds to PD-1 and comprises a VH domain of SEQ ID NO: 11 and a VL domain of SEQ ID NO: 12; Includes.

[0111] In some embodiments, a multispecific antibody (e.g., a bispecific antibody) of the invention comprises: - a first binding site which specifically binds to NRP-1 and comprises a light chain variable domain (VL) sequence of SEQ ID NO: 9 and a heavy chain variable domain (VH) sequence of SEQ ID NO: 10; and a second binding site that specifically binds to PD-1 and comprises a VH domain of SEQ ID NO: 15 and a VL domain of SEQ ID NO: 16; Includes.

[0112] A further object of the present invention relates to a method for treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a multispecific antibody of the present invention comprising at least one binding site that specifically binds to an immune checkpoint molecule and at least one binding site that specifically binds to NRP-1.

[0113] CAR-T cells with suppressed NRP-1 expression: A further object of the present invention relates to a cell population that is engineered to express a chimeric antigen receptor (CAR), wherein expression of NRP-1 in said cells is suppressed.

[0114] In some embodiments, the cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets, including but not limited to T cells, such as tumor infiltrating cells (TILs), CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells, including, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, and mesenchymal stem cells. In some embodiments, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage of using stem cells is that they can be differentiated into other cell types in vitro or can be introduced into a mammal (e.g., a cell donor) where they will engraft in the bone marrow. Methods are known for differentiating CD34 positive cells in vitro into clinically important immune cell types using cytokines such as GM-CSF (granulocyte-macrophage colony-stimulating factor), IFN-γ (interferon-γ) and TNF-α (tumor necrosis factor-α) in vitro (see Inaba et al., J. Exp. Med. 176:1693-1702(1992)).

[0115] In some embodiments, the portion of the CAR (chimeric antigen receptor) of the present invention that comprises an antibody or antibody fragment thereof can exist in various forms, where the antigen binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that includes an scFv.

[0116] In some embodiments, the present invention provides a number of chimeric antigen receptors (CARs) that include an antigen binding domain (e.g., an antibody or antibody fragment, a T cell receptor or a T cell receptor fragment) designed for specific binding to a tumor antigen, e.g., a tumor antigen described herein.

[0117] In some embodiments, the cells (e.g., T cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express the CAR. In some embodiments, the cells (e.g., T cells) are transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR.

[0118] In some embodiments, the antigen binding domain (e.g., scFv) of the CAR of the present invention is encoded by a nucleic acid molecule whose sequence is optimized for expression in mammalian cells. In some embodiments, the entire CAR construct of the present invention is encoded by a nucleic acid molecule whose entire sequence is optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be coded for by various nucleotide sequences. Various codon optimization methods are known in the art, including, for example, those disclosed at least in U.S. Patent Nos. 5,786,464 and 6,114,148.

[0119] In some embodiments, the expression of NRP-1 is suppressed by using an endonuclease. In some embodiments, the expression of NRP-1 is suppressed by using a CRISPR-associated endonuclease. A CRISPR / Cas system for gene editing in eukaryotic cells typically includes (1) a guide RNA molecule (gRNA) that includes a targeting sequence (which can hybridize to a target sequence of genomic DNA) and a sequence that can bind to a Cas, such as a Cas9 enzyme, and (2) a Cas, such as a Cas9 protein. The targeting sequence and the sequence that can bind to a Cas, such as a Cas9 enzyme, can be located on the same molecule or on different molecules. When located on different molecules, each includes a hybridization domain that allows the molecules to associate, for example, through hybridization. Artificial CRISPR / Cas systems that inhibit NRP-1 can be created using techniques known in the art, such as those described in U.S. Patent Publication No. 20140068797, WO 015 / 048577, and Cong (2013) Science 339: 819-823. Other artificial CRISPR / Cas systems that inhibit NRP-1 can also be created, which are known in the art, such as those described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Patent Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359, the contents of which are incorporated herein by reference in their entirety. Such a system for inhibiting NRP-1 can be created, for example, by designing a CRISPR / Cas system to include a gRNA molecule that includes a targeting sequence that hybridizes to an NRP-1 gene sequence. In some embodiments, the gRNA includes a targeting sequence that is fully complementary to 15-25 nucleotides, e.g., 20 nucleotides, of the NRP-1 gene.In some embodiments, 15 to 25 nucleotides, e.g., 20 nucleotides, of the NRP-1 gene are located immediately 5' to a protospacer adjacent motif (PAM) sequence recognized by a Cas protein of a CRISPR / Cas system (e.g., a system herein comprising the Cas9 protein of Streptococcus pyogenes, wherein the PAM sequence comprises NGG, where N can be any of A, T, G, or C).

[0120] In some embodiments, foreign DNA (e.g., DNA encoding a CAR) can be introduced into a cell along with a CRISPR / Cas system.

[0121] In some embodiments, contacting the cells with the endonuclease system occurs ex vivo, hi embodiments, the cells are contacted before, simultaneously with, or after being modified to express a CAR, such as a CAR as described herein.

[0122] In some embodiments, the expression of at least one immune checkpoint protein (e.g., PD-1, CTLA-4) is also inhibited in the cell.

[0123] A further object of the present invention relates to a method for producing a cell expressing a CAR, comprising the steps of: i) introducing into a cell a nucleic acid encoding a CAR; and ii) suppressing the expression of NRP-1 by contacting the cell with an endonuclease system.

[0124] In some embodiments, the method comprises the steps of: i) introducing into a cell a nucleic acid encoding a CAR, and ii) contacting the cell with a Cas protein and at least one guide RNA molecule (gRNA) comprising a sequence that targets the NRP-1 gene and a sequence capable of binding to the Cas protein. In some embodiments, the cell is also contacted with at least one guide RNA molecule comprising a sequence that targets a gene encoding an immune checkpoint protein (e.g., PD-1, CTLA-4, etc.).

[0125] Once the T cell population is obtained, the function of the cells can be evaluated according to any standard method, typically including cytotoxicity assay. The cell surface phenotype of the cells can also be confirmed using appropriate binding pairs. Quantification of the secretion of various cytokines can also be performed. Methods for quantifying the secretion of cytokines in a sample are well known in the art. For example, any immunological method, such as but not limited to ELISA, multiplex strategy, ELISPOT, immunochromatography technology, proteomics method, Western blot, FACS, or radioimmunoassay, can be applicable to the present invention.

[0126] The T cell population obtained by the method of the present invention can find various applications.More particularly, the T cell population is suitable for adoptive immunotherapy.Adoptive immunotherapy is a suitable treatment for any disease or disease state where it has been demonstrated that the elimination of infected or transformed cells is achieved by a specific T cell population.Exemplary diseases, disorders or conditions that can be treated using the T cell population prepared according to the present invention include, for example, infection, such as viral infection, bacterial infection, mycoplasma infection, fungal infection and parasitic infection; and cancer.

[0127] A further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a population of T cells engineered to express a chimeric antigen receptor (CAR), wherein expression of NRP-1 in the cells is suppressed.

[0128] 1. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor in combination with a cancer vaccine: A further object of the present invention relates to a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor in combination with a cancer vaccine.

[0129] In some embodiments, the NRP-1 inhibitor is an anti-NRP-1 antibody derived from the anti-NRP1 antibody YW64.3 described in Mol. Biol. (2007) 366, 815-829 and U.S. Patent Application No. 8378080B1. In particular, the anti-NRP-1 antibody is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); Includes.

[0130] In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9. In some embodiments, the anti-NRP-1 antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 10. In some embodiments, the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO: 9 and a heavy chain variable domain sequence of SEQ ID NO: 10.

[0131] In some embodiments, the NRP-1 inhibitor is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); and an anti-NRP-1 antibody that cross-competes for binding to an NRP-1 isoform with an antibody comprising the following:

[0132] Various substances can be used as antigens in immunogenic or vaccine type compounds or formulations. For example, attenuated and inactivated viral and bacterial pathogens, purified macromolecules, polysaccharides, toxoids, recombinant antigens, organisms containing foreign genes from pathogens, synthetic peptides, polynucleic acids, antibodies, and tumor cells can be used to prepare the cancer vaccines of the present invention. In some embodiments, the antigen is a protein or peptide encoded by DNA or other suitable nucleic acid sequence introduced into cells by transfection, lentiviral or retroviral transduction, minigene introduction, or other suitable procedure. In some embodiments, the antigen is a protein that can be obtained by recombinant DNA technology or by purification from different tissue or cell sources. Typically, the protein has a length of more than 10 amino acids, preferably more than 15 amino acids, and even more preferably more than 20 amino acids, with no theoretical upper limit. Such proteins are not limited to natural proteins, but also include modified proteins or chimeric constructs obtained, for example, by changing selected amino acid sequences or by fusing parts of different proteins. In some embodiments, the antigen is a synthetic peptide. Typically, the synthetic peptide is 3-40 amino acids long, preferably 5-30 amino acids long, and even more preferably 8-20 amino acids long. Synthetic peptides can be obtained by Fmoc biochemical procedures, large-scale synthesis of multiple peptides, recombinant DNA technology, or other suitable procedures. Such peptides are not limited to naturally occurring peptides, but also include modified peptides, post-translationally modified peptides, or chimeric peptides, obtained, for example, by changing or modifying a selected amino acid sequence, or by fusing parts of different proteins.

[0133] In some embodiments, the vaccine composition comprises at least one antigen-presenting cell population presenting a selected antigen. Antigen-presenting cells (or stimulator cells) typically have MHC class I or class II molecules on their surface, and in some embodiments, the antigen-presenting cells themselves are not capable of loading substantially the selected antigen onto MHC class I or class II molecules. Preferably, the antigen-presenting cells are dendritic cells. Suitably, the dendritic cells are autologous dendritic cells that have been flash-pulsed with an antigen of interest (e.g., a peptide). T cell therapy using autologous dendritic cells that have been flash-pulsed with a peptide derived from a tumor-associated antigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278. Thus, in some embodiments, the vaccine composition containing at least one antigen-presenting cell is flash-pulsed or loaded with one or more antigenic peptides. Alternatively, the antigen-presenting cell comprises an expression construct encoding an antigenic peptide. The polynucleotide may be any suitable polynucleotide, which can be transduced into a dendritic cell, preferably, to present the peptide and induce an immune response.

[0134] A further object of the present invention relates to a cancer vaccine comprising an immune adjuvant, wherein the immune adjuvant is an NRP-1 inhibitor, together with one or more cancer antigens to induce an immune response against said one or more cancer antigens.

[0135] Pharmaceutical Compositions: According to the present invention, the active substance is administered to the patient in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier.The pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.For use in administering to a patient, the composition will be formulated for administration to a patient.The composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The use herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. The sterile injectable form of the composition of the present invention may be an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any blended fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutical-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants (e.g., carboxymethylcellulose) or similar dispersants commonly used in formulating pharma- ceutically acceptable dosage forms (e.g., emulsions and suspensions). Other commonly used surfactants (e.g., Tween, Span) and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes. The compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. Diluents useful for oral administration in capsule dosage forms include, for example, lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents may also be added. Alternatively, the compositions of the present invention may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations for each of these areas or organs are easily prepared. For topical application, the compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the compositions may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma- ceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application to the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. A patch may also be used. The compositions of the invention can also be administered by intranasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, which may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, the antibody present in the pharmaceutical composition of the invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for intravenous administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. An exemplary suitable dose range for the antibody in the pharmaceutical composition of the invention is about 1 mg / m. 2 to 500 mg / m 2 The dosage may be between 100 mg and 200 mg. However, these schedules are examples, and optimal schedules and regimens can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition, which must be determined in clinical trials. Pharmaceutical compositions of the invention for injection (e.g., intramuscular, intravenous) can be prepared to contain sterile buffered water (e.g., 1 ml for intramuscular use) and 1 ng to about 100 mg, e.g., about 50 ng to about 30 mg, or more preferably about 5 mg to about 25 mg of the inhibitor of the invention.

[0136] A method for predicting whether a patient suffering from cancer will achieve a response using an immune checkpoint inhibitor: A further object of the present invention relates to a method for predicting whether a patient suffering from cancer will achieve a response with an immune checkpoint inhibitor, comprising: i) determining the expression level of NRP-1 or Semaphorin 3A in a tumor sample from the patient; and ii) comparing the expression level determined in step i) with a predefined reference value; and iii) concluding, if the expression level determined in step i) is lower than the predefined reference value, that the patient will achieve a response with said immune checkpoint inhibitor, or, if the expression level determined in step i) is higher than the predefined reference value, concluding, that the patient will not achieve a response with said immune checkpoint inhibitor.

[0137] Thus, the method is particularly suitable for distinguishing between responders and non-responders. According to the present invention, a responder shows an objective response, and therefore the term does not encompass patients whose cancer has stabilized and whose disease has not progressed after treatment with an immune checkpoint inhibitor. A non-responder or refractory patient includes patients whose cancer does not show regression or improvement after treatment with an immune checkpoint inhibitor. Typically, the characterization of a patient as a responder or non-responder can be performed by reference to a standard or training set. The standard can be a profile of patients known to be responders or non-responders, or alternatively a numerical value. Such a predetermined standard can be provided in any suitable form, for example a printed list or diagram, a computer software program, or other medium.

[0138] In some embodiments, the expression level of NRP-1 or Semaphorin 3A in the tumor tissue sample is determined by immunohistochemistry, for example by contacting the tumor tissue sample with a binding partner (e.g., an antibody) specific for NRP-1 or Semaphorin 3A.

[0139] Immunohistochemistry typically includes the following steps: i) fixation of tumor tissue samples with formalin, ii) embedding of the tumor tissue samples in paraffin, iii) cutting of the tumor tissue samples into sections for staining, iv) incubation of the sections with a binding pair specific for NRP-1 protein, v) rinsing of the sections, vi) incubation of the sections with a secondary antibody, typically biotinylated, and vii) revealing the antigen-antibody complex, typically using an avidin-biotin-peroxidase complex. Thus, the tumor tissue sample is first incubated with a binding pair specific for NRP-1 protein. After washing, the labeled antibody bound to NRP-1 protein is revealed by an appropriate technique, depending on the type of label (e.g. radioactive, fluorescent, or enzymatic) generated by the labeled antibody. Multiple labels may be performed simultaneously. Alternatively, the method of the present invention may use an amplification system (to enhance the staining signal) and a secondary antibody bound to an enzyme molecule. Such conjugated secondary antibodies are commercially available, e.g., from Dako, EnVision systems. Counterstains may also be used, e.g., hematoxylin and eosin, DAPI (diamidinophenylindole), Hoechst, etc. Other staining methods may be accomplished using any suitable method or system, including automated, semi-automated, or manual systems, as would be apparent to one of skill in the art.

[0140] Thus, in some embodiments, the method of the present invention comprises the steps of: i) preparing one or more immunostained tissue section slices obtained by an automated slide staining system by using a binding pair capable of selectively interacting with NRP-1; ii) proceeding with the digitization of the slides of step i) by high resolution scan capture; iii) detecting the tissue section slices on the digital photograph; iv) preparing a size reference grid with uniformly distributed units having the same surface area, the grid being adapted to the size of the tissue section to be analyzed; and v) detecting, quantifying and measuring the intensity or absolute number of stained cells within each unit.

[0141] In some embodiments, the method further comprises determining the expression level of CD8. In some embodiments, the method comprises determining the density of CD8 positive, NRP-1 positive cells in the tumor tissue sample.

[0142] Multiple tissue analysis techniques are particularly useful for quantifying several proteins (e.g., NRP-1 and CD8) in tumor tissue samples. Such techniques should allow the measurement of at least five, or at least ten, or more biomarkers from a single tumor tissue sample. Furthermore, it is advantageous for the technique to be able to preserve the location of the biomarkers and distinguish between the presence of the biomarkers in cancer cells and non-cancerous cells. Such methods include, for example, layered immunohistochemistry (L-IHC), layered expression scanning (LES), or multiple tissue immunoblot (MTI), as taught in U.S. Pat. Nos. 6,602,661; 6,969,615, 7,214,477, and 7,838,222; U.S. Publication No. 2011 / 0306514 (incorporated herein by reference); and Chung & Hewitt, Meth Mol Biol, Prot Blotting Detect, Kurlen & Scofield, eds. 536: 139-148, 2009, each reference teaching that the generation of images of up to 8, 9, 10, 11, or more tissue sections onto layered blotted membranes, papers, filters, and the like may be used. Coated membranes useful for carrying out the L-IHC / MTI process are available from 20 / 20 Gene Systems, Inc. (Rockville, MD).

[0143] In some embodiments, the L-IHC method can be performed on any of a variety of tissue samples, fresh or preserved. Samples include needle biopsies, which were fixed in 10% normal buffered formalin and processed in the pathology department. Standard 5 μm thick tissue sections were cut from the tissue blocks and placed on charged slides, which were used for L-IHC. Thus, L-IHC allows the testing of multiple markers in tissue sections by obtaining copies of molecules transferred from the tissue section to multiple biocompatible coated membranes, essentially creating a copy of the tissue "image". In the case of paraffin sections, the tissue sections are deparaffinized as known in the art, for example, by exposing the sections to xylene or a xylene substitute, such as Neoclear®, and graded ethanol solutions. Sections can be treated with proteinases, such as papain, trypsin, proteinase K, and the like. A laminated membrane substrate, comprising multiple sheets of 10 μm thick coated polymer scaffolds with 0.4 μm diameter holes for the permeation of tissue molecules, such as proteins, through the laminate, is then placed on the tissue section. The membrane substrate is configured such that the movement of liquid and tissue molecules is substantially perpendicular to the membrane surface. The sandwich of the section, membrane, spacer paper, absorbent paper, weights, etc. can be exposed to heat to promote the movement of molecules from the tissue to the laminated membrane. A portion of the tissue proteins is captured on each biocompatible coated laminated membrane (available from 20 / 20 Gene Systems, Inc., Rockville, MD). Each membrane thus contains a copy of the tissue, which can be probed for various biomarkers using standard immunoblot techniques, allowing unlimited amplification of a single marker profile as performed on only one tissue section.Since the amount of protein may be less on membranes in the stack that are further away from the tissue, which may result from, for example, different amounts of molecules in the tissue sample, different mobilities of molecules released from the tissue sample, different binding affinities of molecules to the membrane, length of transfer time, etc., the procedure includes normalizing values, running controls, evaluating the transfer levels of tissue molecules, etc., to correct for changes occurring within the membrane, between membranes (between two parties), and between membranes (between three or more parties), and allows for direct comparison of information within membranes, between membranes (between two parties), and between membranes (between three or more parties). Thus, the total protein per membrane may be determined using any means to reveal bound biotin, for example, by quantifying proteins, for example biotinylated available molecules, for example proteins, using standard reagents and methods, and then exposing the membrane to labeled avidin or streptavidin; protein stains, such as Blot fastStain, Ponceau red, brilliant blue stain, etc., as known in the art.

[0144] In some embodiments, the methods of the present invention use multiple tissue imprinting (MTI) technology to measure biomarkers, where the methods conserve valuable biopsy tissue by allowing for multiple biomarkers, in some cases at least six biomarkers.

[0145] In some embodiments, there are alternative multiple tissue analysis systems that may also be used as part of the present invention. One such technology is the mass spectrometry-based Selected Reaction Monitoring (SRM) assay system ("Liquid Tissue" available from OncoPlexDx, Inc., Rockville, MD). The technology is described in U.S. Patent No. 7,473,532.

[0146] In some embodiments, the methods of the invention utilize a multiplex immunohistochemistry technique developed by GE Global Research (Niskayuna, NY), which is described in U.S. Patent Publication Nos. 2008 / 0118916 and 2008 / 0118934, in which a sequential analysis of a biological sample containing multiple targets is performed, which involves binding a fluorescent probe to the sample, then detecting a signal, then inactivating the probe, followed by binding, detecting, and inactivating the probe to another target, and continuing the process until all targets have been detected.

[0147] In some embodiments, multiple tissue imaging can be performed, and where fluorescence (e.g., fluorophores or quantum dots) is used, the signals can be measured using a multispectral imaging system. Multispectral imaging is a technique in which spectroscopic information at each pixel of an image is collected and the resulting data is analyzed using spectral image processing software. For example, the system can take a series of images at various wavelengths that can be electronically selected in sequence, and then be used with an analysis program designed to handle such data. Thus, the system can obtain quantitative information from multiple dyes simultaneously, even when the dyes' spectra are highly overlapping, or even when they are co-localized, i.e., present at the same point in the sample, but with different spectral curves. Many biological materials autofluoresce, or emit light of lower energy when excited by light of higher energy. This signal can result in images and data with lower contrast. A high-sensitivity camera without multispectral imaging capabilities will only increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging can separate, or separate, the autofluorescence coming from the tissue, thereby increasing the achievable signal-to-noise ratio. Briefly, quantification may be performed by the following steps: i) preparing a tumor tissue microarray (TMA) from a patient; ii) then staining the TMA samples with an anti-antibody with specificity for the NRP-1 protein(s) of interest; iii) further staining the TMA slides with an epithelial cell marker to aid in automated segmentation of tumor and stroma; iv) then scanning the TMA slides using a multispectral imaging system; v) processing the scanned images using automated image analysis software (e.g., PerkinElmer Technology) that allows for detection, quantification, and segmentation of specific tissues through powerful pattern recognition algorithms.Machine learning algorithms are typically pre-trained to segment tumor from stroma and identify labeled cells.

[0148] In some embodiments, the expression level of NRP-1 is determined by determining the amount of mRNA encoding NRP-1. Methods for determining the amount of mRNA are well known in the art. For example, the nucleic acid contained in the sample (e.g., cells or tissues prepared from a patient) is first extracted according to standard methods, for example, using lytic enzymes or chemical solutions, or extracted by nucleic acid binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis, in situ hybridization) and / or amplification (e.g., reverse transcription PCR). Other amplification methods include ligase chain reaction (LCR), transcription-based amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).

[0149] Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but will typically be at least about 80% identical, more preferably 85% identical, and even more preferably 90-95% identical to a homologous region of comparable size. In some embodiments, it will be advantageous to use the nucleic acid in combination with an appropriate means, such as a detectable label, for detection of hybridization.

[0150] Typically, the nucleic acid probe comprises one or more labels, for example to enable detection of the target nucleic acid molecule using the disclosed probe. In various applications, such as in situ hybridization procedures, the nucleic acid probe comprises a label (e.g., a detectable label). A "detectable label" is a molecule or material that can be used to generate a detectable signal that indicates the presence or concentration of a probe (especially a bound or hybridized probe) in a sample. Thus, the labeled nucleic acid molecule provides an indication of the presence or concentration of a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in a sample to which the labeled unique specific nucleic acid molecule is bound or hybridized. The label attached to one or more nucleic acid molecules (e.g., a probe produced by the disclosed method) can be detected either directly or indirectly. The label can be detected by any known or yet to be discovered mechanism, including absorption, emission and / or scattering of photons (including photons of radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency). Detectable labels include colored, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (e.g., enzymes) that convert one substance to another resulting in a detectable difference (e.g., by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of the sample), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

[0151] Particular examples of detectable labels include fluorescent molecules (or fluorescent dyes). Numerous fluorescent dyes are known to those of skill in the art and can be selected, for example, from Life Technologies (formerly Invitrogen), see, e.g., The Handbook-A Guide to Fluorescent Probes and Labeling Technologies. Examples of particular fluorophores that can be attached (e.g., chemically conjugated) to nucleic acid molecules (e.g., unique specific binding regions) are described in Nazarenko et al. No. 5,866,366 to al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3 vinylsulfonyl]phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, brilliant yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylphenyl]naphthalimide ...5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid ethylcoumarin (AMC, Coumarin 120); 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanosine; 4',6-diamidino-2-phenylindole (DAPI); 5',5''dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (disulfor1ic acid);5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC);Eosin and derivatives, such as eosin and eosin isothiocyanate;Erythrosine and derivatives, such as erythrosine B and erythrosine isothiocyanate;Ethidium;Fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q (RITC); 2',7'-difluorofluorescein (Oregon Green®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; phenol red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate, and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine. Rhodamine (ROX), 6-carboxyrhodamine (R6G), Lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates that emit at about 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, Lissamine™, diethylaminocoumarin, chlorotriazinyl fluorescein, naphthofluorescein, 4,7-dichlororhodamine, and xanthene (as disclosed in U.S. Patent No. 5,800,996 to Lee et al.) and their derivatives. Other fluorophores known to those of skill in the art, such as those available from Life Technologies (Invitrogen Inc.;Those available from Molecular Probes (Eugene, Oreg.) can also be used, such as the ALEXA FLUOR® series of dyes (e.g., as described in U.S. Pat. Nos. 5,696,157, 6,130,101, and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, e.g., as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663, and 5,433,896), Cascade Blue (an amine-reactive derivative of sulfonated pyrene described in U.S. Pat. No. 5,132,432), and Marina Blue (U.S. Pat. No. 5,830,912);

[0152] In addition to the fluorescent dyes mentioned above, the fluorescent label can be a fluorescent nanoparticle, e.g., a semiconductor nanocrystal, e.g., QUANTUM DOT™ (obtained, e.g., from Life Technologies, Inc. (QuantumDot, Invitrogen Nanocrystal Technology, Eugene, OR); see also U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649,138). Semiconductor nanocrystals are visible microparticles that have size-dependent optical and / or electrical properties. Irradiation of the semiconductor nanocrystal with a first energy source results in emission of a second energy at a frequency corresponding to the band gap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light or fluorescence of a specific wavelength. Semiconductor nanocrystals with various spectral characteristics are described, for example, in U.S. Pat. No. 6,602,671. Semiconductor nanocrystals can be attached to various biological molecules (e.g., dNTPs and / or nucleic acids) or substrates by techniques described, for example, in Bruchez et al., Science 281:2013-2016, 1998; Chan et al., Science 281:2016-2018, 1998; and U.S. Patent No. 6,274,323. The formation of semiconductor nanocrystals of various compositions is described, for example, in U.S. Patent Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,03 Nos. 8, 6,048,616, 5,990,479, 5,690,807, 5,571,018, 5,505,928, 5,262,357, and U.S. Patent Publication No. 2003 / 0165951 and PCT Publication No. 99 / 26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit different colors of light based on their composition, size, or size and composition.For example, quantum dots that emit at different wavelengths based on size (emission wavelengths of 565 nm, 655 nm, 705 nm, or 800 nm) that are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies, Inc. (Carlsbad, Calif.). Additional labels can include, for example, radioisotopes (e.g., 3 H), metal chelates, such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions, such as Gd3+, and liposomes. Detectable labels that can be used with nucleic acid molecules also include enzymes, such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase, or β-lactamase. Alternatively, enzymes can be used in metallographic detection schemes. For example, the silver in situ hybridization (SISH) procedure includes a metallographic detection scheme for identifying and localizing hybridized genomic target nucleic acid sequences. Metallographic detection methods include the use of enzymes, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive enzyme substrate. The substrate is converted by the enzyme to a redox active substance, which reduces the metal ion, causing it to form a detectable precipitate (see, e.g., U.S. Patent Application Publication No. 2005 / 0100976, PCT Publication No. 2005 / 003777, and U.S. Patent Application Publication No. 2004 / 0265922). Metallographic detection methods also include the use of oxidoreductases (e.g., horseradish peroxidase) in conjunction with water-soluble metal ions, oxidizing agents, and reducing agents, again to form a detectable precipitate (see, e.g., U.S. Patent No. 6,670,113).

[0153] Probes generated using the disclosed methods can be used for detection of nucleic acids, for example, in in situ hybridization procedures (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

[0154] In situ hybridization (ISH) involves contacting a sample (e.g., a cell sample or tissue sample mounted on a slide) containing a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation with a labeled probe capable of specifically hybridizing or specific to the target nucleic acid sequence (e.g., a genomic target nucleic acid sequence). The slide is optionally pretreated, e.g., to remove paraffin or other materials that may interfere with homogeneous hybridization. Both the sample and the probe are treated, e.g., by heating, to denature double-stranded nucleic acids. The probe (formulated in an appropriate hybridization buffer) and the sample are combined under conditions and for a sufficient time to allow hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of the specific label of the chromosomal target is performed using standard techniques.

[0155] For example, biotinylated probes can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For detection of fluorescent dyes, the fluorescent dyes can be detected directly or the sample can be incubated with avidin conjugated to, for example, fluorescein isothiocyanate (FITC). Amplification of the FITC signal can be performed, if necessary, by incubation with a goat anti-avidin antibody conjugated to biotin, washing, and a second incubation with avidin conjugated to FITC. For detection by enzymatic activity, the sample can be incubated, for example, with streptavidin, washed, incubated with alkaline phosphatase conjugated to biotin, washed again, and pre-equilibrated (for example in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, for example, U.S. Pat. No. 4,888,278.

[0156] Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described, for example, in Tanner et al., Am. .1. Pathol. 157:1467-1472, 2000, and U.S. Patent No. 6,942,970. Additional detection methods are provided in US Pat. No. 6,280,929.

[0157] Numerous reagents and detection schemes can be used in conjunction with FISH, CISH and SISH procedures to improve sensitivity, resolution or other desired properties. As discussed above, probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when FISH is performed. Alternatively, probes may be labeled with non-fluorescent molecules, such as haptens (including the following non-limiting examples: biotin, digoxigenin, dinitrophenol, and various oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenone, coumarins, coumarin-based compounds, podophyllotoxins, podophyllotoxin-based compounds, and combinations thereof), ligands, or other indirectly detectable moieties. Such non-fluorescent molecule labeled probes (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., a cell or tissue sample to which the probes bind) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding pair), that is specific for a selected hapten or ligand. The detection reagent may be labeled with a fluorophore (e.g., QUANTUM DOTS®) or with another indirectly detectable moiety, or may be contacted with one or more additional specific binding substances (e.g., a secondary antibody or a specific antibody) that may be labeled with a fluorophore.

[0158] In another example, a probe or specific binding substance (e.g., an antibody, such as a primary antibody, a receptor, or other binding substance) is labeled with an enzyme capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored, or otherwise detectable signal (e.g., as in the deposition of detectable metal particles in SISH). As noted above, the enzyme can be attached directly to the associated probe or detection reagent or indirectly via a linker. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linkers and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006 / 0246524; 2006 / 0246523, and 2007 / 0117153.

[0159] It will be understood by those skilled in the art that by appropriately selecting labeled probe-specific binding substance pairs, multiplex detection schemes can be created that facilitate the detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on one cell or tissue sample, or on more than one cell or tissue sample). For example, a first probe corresponding to a first target sequence may be labeled with a first hapten, such as biotin, while a second probe corresponding to a second target sequence may be labeled with a second hapten, such as dinitrophenol. After exposing the sample to the probe, the bound probe can be detected by contacting the sample with a first specific binding substance (in this case avidin labeled with a first fluorophore, e.g., a first spectrally distinct QUANTUM DOTS® that emits at 585 nm) and a second specific binding substance (in this case an anti-dinitrophenol antibody or antibody fragment labeled with a second fluorophore (e.g., a second spectrally distinct QUANTUM DOTS® that emits at 705 nm)). Additional probe / binding agent pairs can be added in multiplex detection schemes using other spectrally distinct fluorophores. Numerous direct and indirect (one-step, two-step, or more) variations can be envisioned, all of which are suitable in the context of the disclosed probes and assays.

[0160] A probe typically comprises a single-stranded nucleic acid of 10-1000, for example 10-800, more preferably 15-700, typically 20-500 nucleotides in length. A primer is typically a shorter single-stranded nucleic acid of 10-25 nucleotides in length designed to perfectly or nearly perfectly match the target nucleic acid to be amplified. Probes and primers are "specific" for the nucleic acid to which they hybridize, i.e., they hybridize preferably under high stringency hybridization conditions (highest melting temperature Tm, e.g., corresponding to 50% formamide, 5x or 6x SCC, where SCC is 0.15M NaCl, 0.015M sodium citrate).

[0161] The nucleic acid primers or probes used in the above amplification and detection methods may be assembled into a kit. Such a kit includes a universal primer and a molecular probe. A preferred kit also includes components required to determine whether amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting specific sequences.

[0162] In some embodiments, the methods of the present invention comprise the steps of providing total RNA extracted from cumulus cells and, more particularly, subjecting the RNA to amplification and hybridization to specific probes using quantitative or semi-quantitative reverse transcription PCR.

[0163] In some embodiments, the level is determined by DNA chip analysis. Such DNA chips or nucleic acid microarrays consist of various nucleic acid probes chemically attached to a substrate, which may be a microchip, a glass slide, or beads the size of a microsphere. The microchip may be composed of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glass, or nitrocellulose. The probes comprise nucleic acids, such as cDNA or oligonucleotides, which may be from about 10 to about 60 base pairs. To determine the level, a test patient sample, optionally first subjected to reverse transcription, is labeled and contacted with the microarray under hybridization conditions, leading to the formation of a complex with the target nucleic acid that is complementary to the probe sequence attached to the microarray surface. The labeled hybridized complexes can then be detected and quantified or semi-quantified. Labeling can be accomplished by a variety of methods, for example by using radioactive or fluorescent labels. Many variations of microarray hybridization techniques are available to those of skill in the art (see, for example, the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

[0164] In some embodiments, the nCounter® Analysis System is used to detect endogenous gene expression. The basis of the nCounter® Analysis System is a unique code assigned to each nucleic acid target to be assayed (International Patent Application Publication No. WO08 / 124847, U.S. Patent No. 8,415,102, and Geiss et al. Nature Biotechnology. 2008. 26(3): 317-325; the contents of each of which are incorporated herein by reference in their entirety). The code is composed of an ordered series of colored fluorescent spots that create a unique barcode for each target to be assayed. For each DNA or RNA target, a pair of probes is designed: a biotinylated capture probe and a reporter probe with a fluorescent barcode. This system is also referred to herein as the nanoreporter code system. A specific reporter and capture probe is synthesized for each target. The reporter probe may comprise at least one first label attachment region to which one or more light-emitting label monomers are attached, constituting a first signal; at least one second label attachment region, not overlapping with the first label attachment region, to which one or more light-emitting label monomers are attached, constituting a second signal; and a first target-specific sequence. Preferably, each sequence-specific reporter probe comprises a target-specific sequence that can hybridize to no more than one gene, and optionally comprises at least three or at least four label attachment regions, each of which comprises one or more light-emitting label monomers that respectively constitute at least one third signal or at least one fourth signal. The capture probe may comprise a second target-specific sequence; and a first affinity tag. In some embodiments, the capture probe may also comprise one or more label attachment regions. Preferably, the first target-specific sequence of the reporter probe and the second target-specific sequence of the capture probe hybridize to different regions of the same gene to be detected. All reporter and capture probes are pooled into one hybridization mixture, the "probe library."The relative amount of each target in one multiplex hybridization reaction is measured. The method involves contacting a tumor tissue sample with a probe library, such that the presence of a target in the sample creates a probe-pair-target complex. The complex is then purified. More specifically, the sample is combined with the probe library, and hybridization occurs in solution. After hybridization, the three-way hybridized complex (probe pair and target) is purified in a two-step procedure using magnetic beads linked to oligonucleotides complementary to the universal sequences present on the capture probe and reporter probe. This double purification process allows the hybridization reaction to be driven to completion with a large excess of target-specific probes, because they are eventually removed and therefore do not interfere with the binding and imaging of the sample. All post-hybridization steps are robotically operated with a custom liquid handling robot (Prep Station, NanoString Technology, Inc.). The purified reactions are typically deposited by a prep station into individual flow cells of a sample cartridge, bound via capture probes to a streptavidin-coated surface, and subjected to electrophoresis to elongate and immobilize the reporter probes. After processing, the sample cartridge is transferred to a fully automated imaging and data collection device (Digital Analyzer, NanoString Technology, Inc.). Each sample is imaged and the level of target is measured by counting the number of times the code for that target is detected. Approximately 10 mm for each sample. 2Typically 600 fields of view (FOVs) showing the binding surface of the are imaged (1376 x 1024 pixels). Typical image densities are 100-1200 counted reporters per field, depending on the degree of multiplexing, sample input, and total amount of target. Data is output in a simple spreadsheet format listing counts per target, per sample. This system can be used with nanoreporters. Additional disclosure regarding nanoreporters can be found in WO 07 / 076129 and WO 07 / 076132, and U.S. Patent Publication Nos. 2010 / 0015607 and 2010 / 0261026, the contents of which are incorporated herein by reference in their entireties. Additionally, the terms nucleic acid probe and nanoreporter can include rationally designed (e.g., synthetic sequences) as described in International Publication No. WO 2010 / 019826 and U.S. Patent Publication No. 2010 / 0047924, which are incorporated by reference in their entireties.

[0165] The expression level of a gene can be expressed as absolute level or normalized level. Typically, the level is normalized by correcting the absolute level of the gene by comparing its expression with that of a gene that is not relevant for determining the cancer stage of a patient, such as the expression of a constitutively expressed housekeeping gene. Suitable genes for normalization include housekeeping genes, such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1 and TFRC. This normalization allows the comparison of the levels in one sample, for example, between a patient sample and another sample, or between samples from different sources.

[0166] A further object of the present invention relates to a method for treating cancer in a patient in need thereof, comprising: i) determining the expression level of NRP-1 or Semaphorin 3A in a tumor tissue sample obtained from the patient; ii) comparing the expression level determined in step i) with a predefined reference value; and iii) administering an immune checkpoint inhibitor to the patient if the expression level determined in step i) is lower than the predefined reference value.

[0167] The present invention will be further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. EXAMPLES

[0168] overview: Targeting immune checkpoints such as programmed cell death 1 (PD1) improved survival in cancer patients by unblocking exhausted CD8+ T cells and thereby restoring antitumor immune responses. 1、2 However, most patients relapse or are refractory to immune checkpoint blockade therapy. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenic embryonic development. 3、4 NRP1 is also expressed in several immune cell types and is involved in the formation, activation, and termination of the immunological synapse. 5~7 NRP1 impairs antitumor immune responses by modulating the activity of macrophages and regulatory T cells 8~10 Here, we show that NRP1 is recruited to the cytolytic synapse of PD1+CD8+ T cells and interacts with PD-1 to enhance PD-1 activity. In mice, specific deletion of Nrp1 in CD8+ T cells improves spontaneous and anti-PD1 antibody-induced anti-tumor immune responses. Similarly, in human metastatic melanoma, expression of NRP1 in tumor-infiltrating CD8+ T cells predicts poor prognosis in patients treated with anti-PD1 antibodies. Finally, the combination of anti-NRP1 and anti-PD1 antibodies is synergistic in humans, particularly in the anti-tumor response of CD8+ T cells, by increasing T cell receptor signaling in CD8+ T cells at the synapse with tumor cells.

[0169] result: Although PD1 is an important factor in exhaustion, its expression is not sufficient to induce an exhaustion profile in CD8+ T cells. For example, in a mouse model of infection with LCMV clone 13, the majority of antigen-specific CD8+ T cells induced to maintain PD1 expression after antigen withdrawal, but only a small proportion of these CD8+ T cells retained the ability to produce cytokines upon challenge with novel LCMV antigens. 11 This observation suggests the involvement of yet another candidate pair. NRP1 cannot signal autonomously. 12 Since NRP1 is also expressed at the synaptic level in activated T cells, we hypothesized that NRP1 may be involved in the suppressive activity of PD1. In vitro, NRP1 was expressed on mouse CD8+ T cells after activation driven by ovalbumin peptides, and the expression intensity was positively correlated with antigen availability. To investigate the expression of NRP1 on CD8+ T cells in vivo, we studied three antigen-specific immunization models, either acute or sustained. As previously reported, 13、14 However, NRP1 was not expressed on naive CD8+ T cells (data not shown). In contrast, activated specific CD8+ T cells expressed NRP1 after intramuscular adeno-associated virus-ovalbumin (AAV-OVA) immunization, with expression peaking at day 21 post-immunization. NRP1 is a specific anti-OVA receptor in the B16-ovalbumin tumor progression model in mice. 257 It was highly expressed by CD8-positive tumor-infiltrating lymphocytes (Fig. 1) and by specific CD8-positive T cells in an exhaustion model of viral infection with LCMV clone 13 strain compared with infection with LCMV Armstrong strain (Fig. 2).

[0170] To further study the role of NRP1 expression in CD8+ T cells in vivo, we generated a mouse model in which CD8+ T cells were specifically nullified for Nrp1 (CD8Nrp1KO) by crossing Nrp1flox / flox mice with CD8Cre transgenic mice. At steady state, CD8Nrp1 knockout mice had no immunological phenotype, and as expected, CD8+ T cells did not express NRP1 upon activation. In antigen-specific antitumor immune responses, tumor growth was significantly reduced in CD8Nrp1 knockout mice compared to controls (Figure 3). Accordingly, analysis of the tumor immune microenvironment in CD8Nrp1 knockout and control mice showed an increased frequency of CD8+ tumor-infiltrating lymphocytes in CD8Nrp1 knockout mice (Figure 4). These results suggest that expression of NRP1 on CD8+ tumor-infiltrating lymphocytes may be involved in the negative regulation of antitumor immune responses.

[0171] The present inventors previously reported that NRP1 was involved in the immunological synapse between T cells and dendritic cells. 5 We then investigated whether NRP1 could be localized at the synapse between T cells and tumor cells and thus participate in the effector function of CD8+ tumor-infiltrating lymphocytes in this special context. To address this question, we mixed genetically engineered T cell receptor OT1 T cells with the cognate antigen (OVA), and then synthesized NRP1. 257 We developed synapse models between tumor cells (EL4-CFP cells) bearing CD8Nrp1 and between activated CD8+ T cells from CD8Nrp1 knockout mice or littermates and allogeneic tumor cells (A20 cells). Imaging flow cytometry analysis of cell conjugates in these models showed that NRP1 and PD1 were recruited together to the synapse between activated CD8+ T cells and tumor cells (Figures 5-6).

[0172] Clustering and colocalization of PD1 and T cell receptors in response to PD-L1 binding to PD1 characterizes exhausted synapses. 15、16 Since it has been previously reported that NRP1 is important for the induction of low levels of phosphorylated ZAP70 at synaptic junctions, we then investigated whether NRP1 is involved in the recruitment and function of PD1 at synapses. First, by immunofluorescence, we showed in vivo that PD1 and NRP1 were colocalized in CD8-positive tumor-infiltrating lymphocytes from mice (data not shown), and that NRP1 was specifically expressed on human PD1-positive CD8-positive tumor-infiltrating lymphocytes (Figure 7). The interaction between NRP1 and PD1 was demonstrated on activated mouse CD8-positive T cells by in vitro proximity ligation assay (DuoLink) (data not shown). By performing co-immunoprecipitation experiments, we provided additional evidence of this interaction in a protein complex (data not shown). In CD8Nrp1 knockout CD8+ T cells, PD1 was expressed, but its localization in the synapse containing tumor cells was significantly reduced compared to CD8+ T cells derived from wild-type mice (Figure 8). Accordingly, phosphorylated ZAP70 was increased in CD8+ T cells derived from CD8Nrp1 knockout compared to controls in the synapse containing tumor cells (Figure 9). In summary, our data suggest that NRP1 is a counterpart of PD1 that enhances the recruitment and activity of PD1 in the synapse between CD8+ T cells and tumor cells.

[0173] We next investigated whether the role of NRP1 in mouse exhaustion also applies to human CD8+ T cells. Within the human tumor microenvironment, NRP1 expression was found on CD8+ tumor-infiltrating lymphocytes, particularly on PD1+CD8+ T cells, and we identified a subset of PD1+CD8+ tumor-infiltrating lymphocytes with low expression of phosphorylated ZAP70 (phospho-ZAP70 lowNRP1-positive, PD1-positive, CD8-positive tumor-infiltrating lymphocytes) (Figure 10). No patients with homozygous NRP1 mutations have been described, possibly due to the lethality of homozygous NRP1 deletion in utero. 17 However, the inventors were able to identify unique patients with NRP1 haploinsufficiency, caused by a heterozygous deletion of the chromosomal region (10p11.22) that contains the NRP1 gene. 18 NRP1 + / - The patient's CD8+ T cells had increased proliferation and CD25 expression following in vitro activation with Staphylococcal enterotoxin b (SEB) superantigen (Figures 11-12). Furthermore, the increased activation of the patient's CD8+ T cells was synergistic when combined with anti-PD1 antibodies.

[0174] To address this synergistic effect between PD1 and NRP1, we evaluated the in vivo efficacy of anti-PD1 antibody in a B16-ovalbumin tumor-growing mouse model. As previously reported in this model, treatment with anti-PD1 antibody had no effect on overall survival in wild-type mice. In contrast, a significant increase in mouse survival was observed in CD8Nrp1 knockout mice, which was more pronounced when treated with anti-PD1 antibody, indicating a strong synergistic effect (Figure 13).

[0175] To assess the role of NRP1 in human cancer, we next performed a computational study analyzing microarray data from metastatic melanoma cancers treated in clinical trials with anti-PD1 antibody therapy. 19 (Figure 14). According to our hypothesis, low expression of NRP1 in tumors before treatment was associated with improved overall patient survival (p=0.040). Since NRP1 may also be expressed on other cells besides CD8-positive T cells, we then investigated the outcome of 28 metastatic melanoma patients treated with anti-PD1 antibody therapy according to the expression of NRP1 on CD8-positive tumor-infiltrating lymphocytes before the start of treatment. According to our hypothesis, we found that low expression of NRP1 in tumors before treatment was associated with improved overall patient survival (p=0.040). Since NRP1 may also be expressed on other cells besides CD8-positive T cells, we then investigated the outcome of 28 metastatic melanoma patients treated with anti-PD1 antibody therapy according to the expression of NRP1 on CD8-positive tumor-infiltrating lymphocytes before the start of treatment.+ / high Compared with patients with CD8-positive tumor-infiltrating lymphocytes, NRP1 - / low We found the highest complete response rate (data not shown) and a trend towards significantly increased recurrence-free survival in patients with CD8-positive tumor-infiltrating lymphocytes (p=0.042, FIG. 15). In summary, our data demonstrate that NRP1 should be considered as a novel exhaustion agent by enhancing PD1 activity on CD8-positive tumor-infiltrating lymphocytes.

[0176] Finally, we demonstrated that the combination of anti-NRP1 and anti-PD1 antibodies is synergistic in human anti-tumor immune responses. Indeed, in an in vivo synapse model between human activated CD8+ T cells and tumor cells (Raji cells), the combination induced increased expression of phosphorylated ZAP70 (and thus T cell receptor signaling) in CD8+ T cells compared to anti-PD1 antibody alone (Figure 16).

[0177] Consideration: NRP1 has already been implicated in the immune response to tumors by acting as a brake on both innate and adaptive immunity. 8~10 Immune checkpoint therapy has provided many successes for cancer patients 1、2 Unfortunately, most patients relapse or remain refractory, even with combinations of immune checkpoint inhibitors. 20 Our observational data in humans suggest that inhibition of NRP1 may be a potential therapeutic strategy to improve the efficacy of anti-PD1 antibodies. No side effects were reported in experiments in mice evaluating the association of anti-PD1 antibody therapy with safety (data not shown). Furthermore, CD8Nrp1 knockout mice cured of B16-ovalbumin tumor cells with anti-PD1 antibodies did not show any autoimmune or inflammatory phenotypes. This observation indicates that it may be safe to use either drugs that can reduce the expression of NRP1 or antibodies that block both NRP1 and PD1 on CD8 positive T cells.

[0178] Here, we report that specific deletion of Nrp1 on CD8+ T cells dramatically enhances the survival rate of mice bearing B16-ovalbumin tumors, and the possibility of cure with the addition of anti-PD1 antibody therapy. Furthermore, we showed that the combination of anti-NRP1 and anti-PD1 antibodies is synergistic in the anti-tumor immune response of human CD8+ T cells. Thus, our data suggest that a strategy using NRP1-deleted CD8+ CAR-T cells alone or in combination with immune checkpoint inhibitors (e.g., anti-PD1 antibodies) may be a way to improve the efficacy of CAR-T cells. Furthermore, our data suggest that bispecific anti-NRP1 / PD1 antibodies may be a way to improve the efficacy of immune checkpoint inhibitors (e.g., anti-PD1 antibodies).

[0179] In conclusion, we have identified NRP1 as a novel immune checkpoint, which acts through a native mechanism by enhancing the inhibitory effect of PD-1 at the synaptic level, and our data strongly suggest that therapeutic inhibition of NRP1 alone or in combination with immune checkpoint inhibitors (e.g. anti-PD1 antibodies) could efficiently suppress tumor growth in human cancers.

[0180] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure.

[0181] [Table 1] TIFF2024170509000007.tif242165

Claims

1. A method for increasing the amount of tumor-infiltrating CD8-positive T cells in a patient suffering from cancer, comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor.

2. The method of claim 1, wherein the NRP-1 inhibitor is an antibody having binding affinity to NRP-1, in particular an antibody that binds to domain c of NRP-1, an antibody having binding affinity to a region of NRP-1 that binds to semaphorin 3A, or an antibody having binding affinity to an amino acid sequence ranging from amino acid residue 1 to amino acid residue 280 of SEQ ID NO:

1.

3. The method of claim 2, wherein the antibody does not inhibit binding of VEGF to NRP-1.

4. The anti-NRP-1 antibody is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and - a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8) The method of claim 2 , comprising:

5. The method of claim 2, wherein the anti-NRP-1 antibody comprises a light chain variable domain sequence of SEQ ID NO:9 and / or a heavy chain variable domain sequence of SEQ ID NO:

10.

6. The anti-NRP-1 antibody is a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5), and - a heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8) The method of claim 2, wherein the antibody cross-competes for binding to an NRP-1 isoform with an antibody comprising:

7. The method of claim 1, wherein the NRP-1 inhibitor comprises a polypeptide comprising domain c of NRP-1; a polypeptide comprising the transmembrane domain of NRP-1; or a polypeptide comprising an amino acid sequence ranging from amino acid residue 1 to amino acid residue 280 of SEQ ID NO:

1.

8. The method of claim 1 , wherein the NRP-1 inhibitor is an NRP-1 expression inhibitor.

9. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor.

10. 7. The method of claim 6, comprising: i) quantifying the density of CD8 positive T cells in a tumor tissue sample obtained from the patient; ii) comparing the density quantified in step i) with a predetermined reference value; and iii) administering a therapeutically effective amount of an NRP-1 inhibitor to the patient.

11. 1. A method for enhancing the efficacy of an immune checkpoint inhibitor administered to a patient as part of a treatment regimen, the method comprising administering to the patient a pharmacologic effective amount of an NRP-1 inhibitor in combination with the immune checkpoint inhibitor.

12. 1. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective combination of an immune checkpoint inhibitor and an NRP-1 inhibitor, wherein administration of the combination results in enhanced therapeutic efficacy compared to administration of the immune checkpoint inhibitor alone.

13. 10. The method of claim 9, wherein the immune checkpoint inhibitor is an antibody selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-BTLA antibody, and an anti-B7H6 antibody.

14. 14. The method of claim 13, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

15. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a multispecific antibody comprising at least one binding site that specifically binds to PD-1 and at least one binding site that specifically binds to NRP-1.

16. The method of claim 11 , wherein the multispecific antibody is a bispecific antibody.

17. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an NRP-1 inhibitor in combination with a cancer vaccine.

18. 1. A method for predicting whether a patient suffering from cancer will achieve a response with an immune checkpoint inhibitor, the method comprising: i) determining an expression level of NRP-1 or Semaphorin 3A in a tumor sample from the patient; and ii) comparing the expression level determined in step i) with a predetermined reference value; and iii) concluding that the patient will achieve a response with the immune checkpoint inhibitor if the expression level determined in step i) is lower than the predetermined reference value, or concluding that the patient will not achieve a response with the immune checkpoint inhibitor if the expression level determined in step i) is higher than the predetermined reference value.

19. The method of claim 13, further comprising determining the expression level of CD8.

20. 1. A method of treating cancer in a patient in need thereof, comprising: i) determining an expression level of NRP-1 or Semaphorin 3A in a tumor tissue sample obtained from the patient; ii) comparing the expression level determined in step i) with a predetermined reference value; and iii) administering an immune checkpoint inhibitor to the patient if the expression level determined in step i) is lower than the predetermined reference value.

21. A multispecific antibody comprising at least one binding site that specifically binds to PD-1 and at least one binding site that specifically binds to NRP-1.

22. The multispecific antibody of claim 17 which is a bispecific antibody.

23. a light chain variable domain comprising the amino acid sequences of the following complementarity determining regions (CDRs): VL-CDR1 (RASQSISSYLA; SEQ ID NO: 3), VL-CDR2 (GASSRAS; SEQ ID NO: 4), and VL-CDR3 (QQYMSVPIT; SEQ ID NO: 5); A heavy chain variable domain comprising the following CDR amino acid sequences: VH-CDR1 (GFSFSSEPIS; SEQ ID NO: 6), VH-CDR2 (SSITGKNGYTYYADSVKG; SEQ ID NO: 7), and VH-CDR3 (WGKKVYGMDV; SEQ ID NO: 8); 18. The multispecific antibody of claim 17, comprising a first binding site that specifically binds to NRP-1, comprising:

24. 18. The multispecific antibody of claim 17, comprising a first binding site that specifically binds to NRP-1, the first binding site comprising the light chain variable domain (VL) sequence of SEQ ID NO: 9 and the heavy chain variable domain (VH) sequence of SEQ ID NO:

10.

25. 18. The multispecific antibody of claim 17, which specifically binds to PD-1 and comprises a second binding site comprising a VH domain of SEQ ID NO:11 and a VL domain of SEQ ID NO:

12.

26. 18. The multispecific antibody of claim 17, which specifically binds to PD-1 and comprises a second binding site comprising a VH domain of SEQ ID NO: 15 and a VL domain of SEQ ID NO:

16.

27. a first binding site which specifically binds to NRP-1 and comprises a light chain variable domain (VL) sequence of SEQ ID NO: 9 and a heavy chain variable domain (VH) sequence of SEQ ID NO: 10; and a second binding site which specifically binds to PD-1 and comprises a VH domain of SEQ ID NO: 11 and a VL domain of SEQ ID NO: 12; 18. The multispecific antibody of claim 17 , comprising:

28. a first binding site which specifically binds to NRP-1 and comprises a light chain variable domain (VL) sequence of SEQ ID NO: 9 and a heavy chain variable domain (VH) sequence of SEQ ID NO: 10; and a second binding site which specifically binds to PD-1 and comprises a VH domain of SEQ ID NO: 15 and a VL domain of SEQ ID NO: 16; 18. The multispecific antibody of claim 17 , comprising:

29. 18. The multispecific antibody of claim 17 for use in the treatment of cancer.

30. A cell population that has been engineered to express a chimeric antigen receptor (CAR), wherein expression of NRP-1 in the cells is suppressed.

31. 22. The cell population of claim 21, wherein the cells are selected from the group consisting of tumor infiltrating cells (TILs), T cells, such as CD4 positive T cells or CD8 positive T cells, and stem cells.

32. The cell population of claim 21, wherein expression of at least one immune checkpoint protein is also suppressed.

33. A method for producing a cell expressing a CAR, comprising the steps of: i) introducing a nucleic acid encoding a CAR into a cell; and ii) contacting the cell with an endonuclease system to suppress expression of NRP-1.

34. 25. The method of claim 24, comprising the steps of: i) introducing into a cell a nucleic acid encoding a CAR; and ii) contacting the cell with a Cas protein and at least one guide RNA molecule (gRNA) comprising a sequence that targets the NRP-1 gene and a sequence capable of binding to the Cas protein.

35. 26. The method of claim 25, further comprising contacting the cell with at least one guide RNA molecule comprising a sequence that targets a gene encoding an immune checkpoint protein (e.g., PD-1, CTLA-4, etc.).

36. 22. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the T cell population of claim 21.

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