Methods of treating cancer using antibodies and molecules that bind to BTN1A1 or BTN1A1-ligands

Abstract

The present invention provides methods of treating cancer using antibodies and molecules that bind to BTN1A1 or BTN1A1-ligands. The present invention provides methods of treating cancer using a molecule having an antigen binding fragment that immunospecifically binds to BTN1A1 or a BTN1A1 ligand, such as an anti-BTN1A1 antibody or an anti-BTN1A1 ligand antibody. The present invention also provides a BTN1A1 ligand, such as a galectin-1 (galectin-1), a galectin-9 (galectin-9), a neuropilin-2 (Neuropilin-2), and a B-and T-lymphocyte attenuating protein, for example, a BTN1A1 ligand, for example, a Galectin-1 (Galectin-1), a Galectin-9 (Galectin-9), a Galectin-9 (Galectin-9), a Galectin-2 (

Description

Cross-reference to related applications

[0001] This application is a divisional application of Chinese Patent Application No. 201880051242.X, filed February 6, 2020, entitled "Method for Treating Cancer Using Antibodies and Molecules Binding to BTN1A1 or BTN1A1-Ligands". This application claims priority to U.S. Provisional Application No. 62 / 516,071, filed June 6, 2017, the disclosure of which is incorporated herein by reference in its entirety. References to sequence lists

[0002] This application is submitted in computer-readable form (CRF) of a sequence list named 13532-020-228_st25.txt, created on June 4, 2018, and is 40,325 bytes in size; the entire contents of which are incorporated herein by reference. 1. Technical Field

[0003] This invention relates generally to the fields of cancer immunology and molecular biology. It provides methods for treating cancer using anti-BTN1A1 antibodies or anti-BTN1A1-ligand antibodies, or other molecules having antigen-binding fragments that specifically bind to BTN1A1 or BTNLA1 ligands. In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1-ligand antibody can disrupt the BTN1A1-BTN1A1 ligand interaction. In some embodiments, the anti-BTN1A1 ligand antibody includes anti-galactolectin 1 (GAL-1) antibody, anti-galactolectin 9 (GAL-9) antibody, anti-neuropiliin-2 (NRP-2) antibody, or anti-B- and T-lymphocyte attenuating protein (BTLA) antibody. 2. Background Technology

[0004] The immune systems of humans and other mammals protect them from infection and disease. Numerous stimulant and inhibitory ligands and receptors provide a tightly controlled system that maximizes the immune response against infection while limiting autoimmunity. Recently, therapeutic agents that modulate immune responses, such as anti-PD1 or anti-PDL1 antibodies, have been found to be effective in the treatment of certain cancers. However, the development of new therapeutic agents that safely and effectively treat diseases by modulating the immune system remains urgently needed, particularly for cancers resistant or refractory to anti-PD1 or anti-PDL1 therapy. The methods described in this article address these needs and offer other relevant advantages. 3. Summary of the Invention

[0005] On one hand, the present invention provides a molecule comprising an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of BTN1A1 ligands, such as galactolectin-1 (GAL-1), galactolectin-9 (GAL-9), NRP-2 (Nrp-2), or B- and T-lymphocyte attenuating proteins (BTLA), to BTN1A1.

[0006] In some embodiments, the antigen-binding fragment specifically binds to BTN1A1, and the molecule inhibits the binding of BTN1A1 to GAL-1.

[0007] In some embodiments, the antigen-binding fragment specifically binds to BTN1A1, and the molecule inhibits the binding of BTN1A1 to GAL-9.

[0008] In some embodiments, the antigen-binding fragment immune-specifically binds to BTN1A1, and the molecule inhibits the binding of BTN1A1 to NRP-2.

[0009] In some embodiments, the antigen-binding fragment specifically binds to BTN1A1, and the molecule inhibits the binding of BTN1A1 to BTLA.

[0010] In some embodiments, the antigen-binding fragment immune-specifically binds to BTN1A1, and the molecule inhibits two or more BTN1A1 ligands, such as GAL-1, GAL-9, NRP-2, or BTLA.

[0011] In some implementations, the antigen-binding fragment immune-specifically binds to the extracellular domain (ECD) of BTN1A1.

[0012] On the other hand, the present invention provides a molecule comprising an antigen-binding fragment of an immune-specific BTN1A1 ligand selected from GAL-1, GAL-9, NRP-2 and BTLA, said molecule inhibiting the binding of the BTN1A1 ligand to BTN1A1.

[0013] In some embodiments, the antigen-binding fragment specifically binds to GAL-1, and the molecule inhibits the binding of GAL-1 to BTN1A1.

[0014] In some embodiments, the antigen-binding fragment specifically binds to GAL-9, and the molecule inhibits the binding of GAL-9 to BTN1A1.

[0015] In some embodiments, the antigen-binding fragment immune-specifically binds to NRP-2, and the molecule inhibits the binding of NRP-2 to BTN1A1.

[0016] In some embodiments, the antigen-binding fragment specifically binds to BTLA, and the molecule inhibits the binding of BTLA to BTN1A1.

[0017] In some embodiments, the molecule modulates the activity or signal transduction of BTN1A1, or modulates the activity or signal transduction of BTN1A1 and its ligand complex, such as GAL-1, GAL-9, NRP-2, or BTLA.

[0018] In some embodiments, the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, with an IC50 of no more than 1 μM.

[0019] In some embodiments, the molecular inhibition of the binding of the BTN1A1 ligand to BTN1A1 has an IC50 of no more than 500 nM, no more than 400 nM, no more than 300 nM, no more than 200 nM, no more than 100 nM, no more than 50 nM, no more than 10 nM, or no more than 5 nM.

[0020] In some implementations, the molecule can modulate T cell activity.

[0021] In some implementations, the T cells are CD8+ cells.

[0022] In some implementations, the molecule can increase T cell activation or T cell proliferation.

[0023] In some implementations, the molecule can inhibit T cell apoptosis.

[0024] In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1.

[0025] In some embodiments, the antigen-binding fragment preferentially binds to the dimer BTN1A1 relative to the monomer BTN1A1.

[0026] In some embodiments, the antigen-binding fragment specifically binds to BTN1A1 or the BTN1A1 ligand with a dissociation constant (KD) of no more than 1 μM.

[0027] In some embodiments, the antigen-binding fragment specifically binds to BTN1A1 or the BTN1A1 ligand with a dissociation constant (KD) of no more than 500 nM, no more than 400 nM, no more than 300 nM, no more than 200 nM, no more than 100 nM, no more than 50 nM, no more than 10 nM, or no more than 5 nM.

[0028] In some embodiments, the KD of the antigen-binding fragment for immune-specific binding to BTN1A1 or a BTN1A1 ligand is less than or equal to the KD of the BTN1A1-GAL-1 interaction, BTN1A1-GAL-9 interaction, BTN1A1-NRP2 interaction, or BTN1A1-BTLA interaction. In some embodiments, the KD of the antigen-binding fragment for immune-specific binding to BTN1A1 or a BTN1A1 ligand is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold lower than the KD of the BTN1A1-GAL-1 interaction, BTN1A1-GAL-9 interaction, BTN1A1-NRP2 interaction, or BTN1A1-BTLA interaction.

[0029] In some embodiments, the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, with an IC50 of no more than 1 μM.

[0030] In some embodiments, the molecular inhibition of the binding of the BTN1A1 ligand to BTN1A1 has an IC50 of no more than 500 nM, no more than 400 nM, no more than 300 nM, no more than 200 nM, no more than 100 nM, no more than 50 nM, no more than 10 nM, or no more than 5 nM.

[0031] In some implementations, the inhibition of BTN1A1 binding, BTN1A1 ligand binding, or BTN1A1 ligand binding is analyzed by co-immunoprecipitation (co-IP), surface plasmon resonance (SPR) assay, β-galactosidase complementation, or biolayer interference (BLI) measurement.

[0032] In some implementations, the molecule is an antibody.

[0033] In some implementations, the molecule is a monoclonal antibody.

[0034] In some implementations, the antibody is a human antibody or a humanized antibody.

[0035] In some implementations, the antibody is IgG, IgM, or IgA.

[0036] In some embodiments, the molecule is Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or a single-domain antibody.

[0037] In some implementations, the molecule is generated through recombination.

[0038] On the other hand, the present invention provides a pharmaceutical composition comprising the provided molecule and a pharmaceutically acceptable carrier.

[0039] In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0040] On the other hand, the present invention provides a method for activating T cells, comprising contacting the T cells with an effective amount of a molecule provided by the present invention, thereby activating the T cells by inhibiting the binding of the BTN1A1 ligand to BTN1A1.

[0041] On the other hand, the present invention provides a method for inhibiting the binding of BTN1A1 ligand to BTN1A1 expressed on T cells, comprising contacting the T cells with an effective amount of the molecule provided by the present invention, thereby inhibiting the binding of the BTN1A1 ligand to the T cells.

[0042] In some implementations, the T cells are CD8+ cells.

[0043] In some implementations, T cell activation includes (i) increasing T cell proliferation, (ii) reducing T cell apoptosis, or (iii) increasing cytokine production.

[0044] In some implementations, the cytokine is IFNγ or IL-2.

[0045] On the other hand, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a molecule or pharmaceutical composition provided by the present invention, wherein the molecule inhibits the binding of a BTN1A1 ligand to BTN1A1 in the subject.

[0046] On the other hand, the present invention provides a method for inhibiting the binding of BTN1A1 ligand to BTN1A1, wherein a therapeutically effective amount of the molecule or pharmaceutical composition provided by the present invention is administered to a subject, wherein the molecule inhibits the binding of BTN1A1 ligand to BTN1A1 in the subject.

[0047] In some implementations, the method further includes administering high-dose radiation therapy to the patient.

[0048] In some implementations, the cancer may include lung cancer, prostate cancer, pancreatic cancer, ovarian cancer, liver cancer, head and neck cancer, breast cancer, and stomach cancer.

[0049] In some implementations, the cancer may include lung cancer.

[0050] In some implementations, the lung cancer is non-small cell lung cancer (NSCLC).

[0051] In some implementations, the NSCLC is a scaly NSCLC.

[0052] In some implementations, the cancer is a cancer that is resistant to or refractory to anti-PD-1 therapy or anti-PD-L1 therapy.

[0053] In some implementations, the cancer is breast cancer or lung cancer.

[0054] In some implementations, the cancer is breast cancer or Lewis lung cancer.

[0055] In some embodiments, the molecule is not STC810 ​​as described in International Patent Application No. PCT / US16 / 64436, or a CDR, VH, or VL amino acid sequence containing STC810. 4. Brief description of the attached drawings

[0056] The following drawings form part of this specification and are included to further illustrate certain embodiments of the invention. A better understanding of the invention can be achieved by referring to one or more of these drawings, in conjunction with the detailed description of the specific embodiments given herein.

[0057] Figure 1 - Identification of galactolectin-1, galactolectin-2 and neurociliamin-2 as BIN1A1 conjugates. Figure 1 Images are shown illustrating the results of an exemplary property membrane protein array experiment that reconfirms galactolectin-1 (GAL-1, “LGALSL”), galactolectin-2 (GAL-2, “LGALS9”), and neurociliacin-2 (NRP-2) as ligands for BTN1A1. Expression vectors for the specified proteins were repeatedly spotted on two slides (“rep1”, “rep2”) and transfected inversely into HEK293T cells. The transfected cells were then fixed and detected individually with CTLA-4-Fc, BTN1A1-2NQ-Fc (unglycosylated), BTN1A-Fc (glycosylated), or a secondary antibody (cells expressing the CD86 / ZsGreen1 positive control vector). Fluorescence imaging was used to detect the binding of the probe proteins to the expressed proteins after the addition of fluorescently labeled secondary antibodies.

[0058] Figure 2A -C- Immunoprecipitation confirmed that GAL-1 and GAL-9 are BTN1A1 ligands. Figure 2A A schematic diagram of the BTN1A1 and BTN1A1-ligand (GAL-1 or GAL-9) protein constructs used for immunoprecipitation is shown. BTN1A1 includes an extracellular domain (ECD), a transmembrane domain (TM), a cytoplasmic protein domain (CPD), and a Flag-tag. The BTN1A1-ligand includes Myc- and Flag-tags. Figure 2BA diagram illustrating the immunoprecipitation assay is shown. BTN1A1 or BTN1A1-ligand is pulled from HEK293T cell lysates using beads coated with anti-BTN1A1 or anti-Myc antibodies. Figure 2C Images of Western blotting after immunoprecipitation are shown. An asterisk (*) indicates a BTN1A1 band or a BTN1A1-ligand band (**).

[0059] Figure 3A -D- Analysis of BTN1A1-GAL-1 interaction via surface plasmon resonance (SPR). Figure 3A -D shows a sensor map of an exemplary SPR measurement. The sensor chip is equipped with immobilized wild-type BTN1Al-Fc (…). Figure 3A ), non-glycosylated BTN1A1-2NQ-Fc ( Figure 3B ), glycosylated wild-type BTN2A1 ( Figure 3C ) or glycosylated wild-type BTN3A2 ( Figure 3D Inject GAL-1 protein.

[0060] Figure 4A and 4B - Identification of BTLA as a ligand for BTN1A1. Figure 4A A schematic diagram illustrating the β-galactosidase (β-Gal) complementation assay is shown, in which β-Gal is divided into an enzyme donor (ED) and an enzyme acceptor (EA). The interaction between the ED fusion protein and the EA fusion protein leads to the reconstruction of functional β-Gal detectable using a luminescent β-Gal substrate. Figure 4B A bar graph illustrating the results of an exemplary β-galactosidase (β-Gal) complementation assay is shown.

[0061] Figure 5 - BTLA was validated as a ligand for BTN1A1 by immunoprecipitation. Figure 5 Exemplary Western blot results are shown after immunoprecipitation of BTN1A1-Flag or BTLA-Myc-Flag with anti-Myc antibody or anti-BTN1A1 antibody (STC810).

[0062] Figure 6A and 6B - Analysis of BTN1A1-BTLA interaction via surface plasmon resonance (SPR). Figure 6A The sensor map showing the SPR analysis shows that GAL-1, BTLA, or control proteins (control 1, control 2, or control 3) were injected at a single concentration of 3.2 μM onto a sensor chip immobilized with glycosylated wild-type BTN1Al-Fc. Figure 6BThe sensor plot of the SPR analysis is shown, in which BTLA is injected at a specified concentration onto a sensor chip with immobilized glycosylated wild-type BTN1A1-Fc.

[0063] Figure 7 - The interaction between BTN1A1 and BTLA was analyzed using biolayer interferometry (BLI). Figure 7 The sensor map of the BLI experiment is shown, in which soluble BTLA is contacted with immobilized BTN1A-Fc at a specified concentration. 5. Detailed Explanation

[0064] The B7 family of co-stimulatory molecules can drive the activation and inhibition of immune cells. A related molecular family—buryrophilins—also possesses immunomodulatory functions similar to those of B7 family members. Butyrophilin, subfamily 1, member A1 (“BTN1A1”) is a type I membrane glycoprotein and a major component of the milk fat globule membrane, exhibiting structural similarities to the B7 family. BTN1A1 is known to be a major protein regulating the formation of fat droplets in milk (Ogget et al. PNAS, 101(27): 10084-10089(2004)). BTN1A1 is expressed in immune cells, including T cells. Treatment with recombinant BTN1A1 has been found to inhibit T cell activation and protect against EAE in animal models (Stefferl et al., J. Immunol. 165(5): 2859-65(2000)).

[0065] BTN1A1 is also specifically and highly expressed in cancer cells. BTN1A1 expressed in cancer cells is usually glycosylated. BTN1A1 expression can be used to aid in cancer diagnosis and assess the effectiveness of cancer treatments.

[0066] This disclosure is based, at least in part, on the discovery that Gal-1, Gal-9, NRP-2, and BTLA can act as ligands for BTN1A1. See, for example, Examples 1 and 2. Without being limited to any particular theory, it is believed that inhibiting the formation of complexes of GAL-1, GAL-9, NRP-2, or BTLA with BTN1A1, including disrupting already formed complexes of BTN1A1 with BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA), can modulate BTLA activity or signaling. It is also believed that such modulation of BTLA activity or signaling can activate T cells (e.g., CD8+ T cells), for example, by promoting T cell proliferation, inhibiting T cell apoptosis, or inducing cytokine secretion (e.g., IFNγ or IL2). T cell activation can lead to anticancer immune responses useful for the treatment or prevention of cancer.

[0067] This invention provides methods for treating cancer using anti-BTN1A1 antibodies, anti-BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies, and other molecules capable of immune-specifically binding to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) and inhibiting the BTN1A-BTN1 ligand complex. Methods for diagnosing cancer and selecting patients are also provided using such anti-BTN1A1 antibodies or anti-BTN1A1-ligand antibodies and other molecules capable of immune-specifically binding to BTN1A1 or BTN1A1 ligands.

[0068] 5.1. Definition Unless otherwise stated, the terms “a,” “an,” and “described” as used herein refer to one or more of the grammatical objects of the article. For example, an antibody refers to one or more antibodies.

[0069] As used herein, unless otherwise stated, the terms "mammoplasmin, subfamily 1, member A1" or "BTN1A1" refer to BTN1A1 of any vertebrate origin, including mammals such as primates (e.g., humans, cynomolgus monkeys), dogs, and rodents (e.g., mice and rats). Unless otherwise stated, BTN1A1 also includes various BTN1A1 isotypes, associated BTN1A1 polypeptides (including their SNP variants), and various modified forms of BTN1A1, including but not limited to phosphorylated BTN1A1, glycosylated BTN1A1, and ubiquitinated BTN1A1. Glycosylated BTN1A1 as used herein includes BTN1A1 with N55, N215, and / or N449 glycosylation.

[0070] Below is an exemplary human BTN1A1 amino acid sequence (BC096314.1GI: 64654887), where potential glycosylation sites are indicated by bold and underline:

[0071] The following is an example of the coding nucleic acid sequence for human BTN1A1 (BC096314GI: 64654887):

[0072]

[0073]

[0074] The following provides an exemplary amino acid sequence of an exemplary dimer BTN1A1 extracellular domain construct (BTN1A1-ECD-Fc).

[0075]

[0076]

[0077] The following provides an exemplary amino acid sequence of an exemplary monomeric BTN1A1 extracellular domain construct (BTN1A1-His6).

[0078]

[0079] The following is an exemplary mouse BTN1A1 amino acid sequence (GenBank: AAH11497.1), where potential glycosylation sites are indicated in bold and underline:

[0080] The following is an exemplary coding nucleic acid sequence for mouse BTN1A1 (GenBank: BC011497.1):

[0081]

[0082]

[0083] As used herein, unless otherwise stated, the terms “galactolectin-1,” “GAL-1,” “GAL1,” “GBP,” or “LGALS1” include polypeptides from any vertebrate source (“polypeptide” and “protein” are used interchangeably herein), including any natural polypeptide, unless otherwise stated. In some embodiments, the term “related GAL-1 polypeptide” includes its SNP variants. The term “GAL-1” also includes “full-length,” untreated GAL-1, and any form of GAL-1 produced by cellular treatment. NCBI reference sequence NP_002296 provides an exemplary human GAL-1 amino acid sequence. NCBI reference sequence NM-002305 provides an exemplary human GAL-1 nucleic acid sequence (mRNA).

[0084] Below is an example amino acid sequence of human GAL-1 (NCBI reference sequence NP_002296).

[0085]

[0086] Below is an example nucleic acid sequence for human GAL-1 (NCBI reference sequence NM_002305 (coding sequence)).

[0087]

[0088] As used herein, unless otherwise stated, the terms “galactolectin-9,” “HUAT,” “GAL-9,” “GAL9,” “LGALS9,” or “LGALS9A” include polypeptides (“polypeptide” and “protein” are used interchangeably herein) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (crab-eating macaques), dogs, and rodents (e.g., mice and rats)), including any native polypeptide unless otherwise stated. In some embodiments, the term includes “related GAL-9 polypeptide,” including its SNP variants. The term “GAL-9” also includes “full-length,” untreated GAL-9, and any form of GAL-9 produced by cellular treatment. NCBI reference sequence NP_001317092 An exemplary human GAL-9 amino acid sequence is provided. The NCBI reference sequence NM_002308 provides an exemplary human GAL-9 nucleic acid sequence (mRNA).

[0089] Below is an example amino acid sequence of human GAL-9 (NCBI reference sequence NP_001317092).

[0090]

[0091] Below is an example nucleic acid sequence for human Gal-9 (NCBI reference sequence NM_002308 (coding sequence)).

[0092]

[0093] As used herein, unless otherwise stated, the terms “neurociliin-2,” “NRP-2,” “NRP2,” “NP2,” “PR02714,” or “VEGF165R2” include polypeptides from any vertebrate source (including mammals such as primates (e.g., humans and cynomolgus monkeys (crab-eating macaques)), dogs, and rodents (e.g., mice and rats)) (“polypeptide” and “protein” are used interchangeably herein), including any natural polypeptide unless otherwise stated. In some embodiments, the term includes “related NRP-2 polypeptide,” including its SNP variants. The term “NRP-2” also includes “full-length,” untreated NRP-2, as well as any form of NRP-2 produced by cellular treatment. NCBI reference sequence NP_003863 provides an exemplary human NRP-2 amino acid sequence. NCBI reference sequence NM_003872 provides an exemplary human NRP-2 nucleic acid sequence (mRNA).

[0094] Below is an example amino acid sequence of human NRP-2 (NCBI reference sequence NP_003863).

[0095]

[0096] Below is an example nucleic acid sequence for human GAL-1 (NCBI reference sequence NM_003872 (coding sequence)).

[0097]

[0098]

[0099] As used herein, unless otherwise stated, the terms “B- and T-lymphocyte attenuating proteins” or “BTLA” refer to BTLA from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (crab-eating macaques)), dogs, and rodents (e.g., mice and rats). Unless otherwise stated, BTLA also includes various BTLA isotypes, associated BTLA polypeptides, including their SNP variants, and various modified forms of BTLA, including but not limited to phosphorylated BTLA, glycosylated BTLA, and ubiquitinated BTLA.

[0100] Below is an exemplary amino acid sequence of human BTLA, where N-linked glycosylation sites are bolded and underlined (N75, N94, and NL10):

[0101] The following is an example nucleic acid sequence of human BTLA (NCBI reference sequence NM_001085357.1 (coding sequence)):

[0102]

[0103] As used herein, unless otherwise stated, the terms “programmed cell death 1,” “programmed cell death 1,” “protein PD-1,” “PD-1,” “PD-1 polypeptide,” or “PD1” include polypeptides from any vertebrate source (including mammals such as primates (e.g., humans and cynomolgus monkeys (crab-eating macaques)), dogs, and rodents (e.g., mice and rats)) (the terms “polypeptide” and “protein” are used interchangeably herein), including any natural polypeptide, unless otherwise stated. In some embodiments, the term includes “related PD-1 polypeptide,” including its SNP variants. The term “PD-1” also includes “full-length,” untreated PD-1, and any form of PD-1 produced by cell treatment. An exemplary human PD-L1 amino acid sequence is provided in NCBI reference sequence NP_005009.2. GenBank TM Accession number L27440.1 provides an exemplary human PD-1 nucleic acid sequence.

[0104] As used herein, unless otherwise stated, the term "anti-PD-1 therapy" includes any PD-1 inhibitor. In some embodiments, anti-PD-1 therapy may include an anti-PD-1 antibody or an antigen-binding fragment thereof, an inhibitory nucleic acid, or a soluble PD-1 ligand (e.g., soluble PD-L1), or a fusion protein thereof (e.g., Fc-fusion protein). In some embodiments, anti-PD-1 therapy includes nivolumab (opdivo), pembrolizumab (Keytruda), pidilizumab, AMP-514, or AMP-224.

[0105] In some implementations, anti-PD-1 therapy includes nivolumab (CAS Registry No.: 946414-94-4). Nivolumab is also known as MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US8008, 449 and WO2002 / 121168.

[0106] In some implementations, anti-PD-1 therapy includes pembrolizumab. Pembrolizumab is also known as Keytruda®, lambolizumab, Merck 3745, MK-3475, or SCH-900475. Pembrolizumab is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab has been published, for example, in the New England Journal of Medicine 369(2): 134-44, WO2002 / 114335, and US8354509, by Hamid, O. et al.

[0107] In some implementations, the anti-PD-1 treatment is pidilizumab. Pidilizumab, also known as CT-011 (CureTech), is a humanized IgG1 monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2002 / 101611.

[0108] In some implementations, anti-PD-1 therapy includes the use of anti-PD-1 antibodies as provided in international application PCT / US20126 / 64394.

[0109] Other anti-PD1 antibodies that can be used as anti-PD1 therapy are disclosed in US8,609,089, US2010028330 and / or US20120114649.

[0110] In some implementations, anti-PD-1 therapy includes the fusion protein AMP514 (Amplimmune). AMP-224, also known as B7-DCIg, is disclosed, for example, in WO2010 / 027827 and WO2011 / 066342. AMP-224 is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1.

[0111] In some embodiments, the anti-PD-1 therapy includes an immunoadhesin (e.g., an immunoadhesin comprising the extracellular portion or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the anti-PD-1 therapy includes the fusion protein AMP-224 (an Fc fusion of PD-L2).

[0112] As used herein, unless otherwise stated, the terms “programmed death-1 ligand 1,” “programmed cell death-1 ligand 1,” “protein PD-L1,” “PD-L1,” “PD-L1 polypeptide,” or “PD1-L1” encompass polypeptides (“polypeptide” and “protein” are used interchangeably herein), including any naturally occurring polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), dogs, and rodents (e.g., mice and rats), unless otherwise stated. In some embodiments, the term includes “associated PD-L1 polypeptide,” including its SNP variants.30 The term “PD-L1” also encompasses “full-length,” unprocessed PD-L1, as well as any processed form of PD-L1 produced in cells. NCBI reference sequence NP_054862.1.2 provides an exemplary human PD-L1 amino acid sequence. GenBank™ registry number NM_014143 provides an exemplary human PD-1 nucleic acid sequence.

[0113] As used herein, unless otherwise stated, the term "anti-PD-L1 therapy" encompasses any inhibitor of PD-L1. In some embodiments, an anti-PD-1 therapy may comprise an anti-PD-L1 antibody or an antigen-binding fragment thereof, an inhibitory nucleic acid, or a soluble PD-L1 ligand (e.g., soluble PD-1), or a fusion protein thereof (e.g., an Fc-fusion protein). In some embodiments, the anti-PD-L1 therapy includes YW243.55.S70, MPD13280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0114] In some implementations, the anti-PD-L1 therapy includes MDX-1105. MDX-1105 is also known as BMS-936559. See, for example, WO2007 / 005874.

[0115] In some embodiments, the PD-L1 therapy includes antibody YW243.55.S70, such as that described in WO2010 / 077634 (the heavy chain and light chain variable region sequences are shown in SEQ ID NO: 20 and 21, respectively).

[0116] In some embodiments, the PD-L1 therapy includes MDPL3280A (Genentech / Roche). MDPL3280A is a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies targeting PD-L1 are disclosed, for example, in U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906.

[0117] In some embodiments, the anti-PD-L1 therapy includes the antibody MSB0010718C (MerckSerono). MSB0010718C is also known as A09-246-2.

[0118] In some embodiments, the anti-PD-L1 therapy includes MDPL3280A (Genentech / Roche), a human Fc-optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed, for example, in U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906.

[0119] In some embodiments, the anti-PD-L1 therapy includes antibodies provided in international applications No. PCT / US2016 / 024691 and No. PCT / US2017 / 024027, disclosed in WO2016 / 160792A1.

[0120] As used herein, unless otherwise stated, the term "antibody" refers to a B-cell polypeptide product within the immunoglobulin (or "Ig") class of polypeptides capable of binding to a specific molecular antigen, consisting of two identical polypeptide chains paired, each pair having a heavy chain (approximately 50-70 kDa) and a light chain (approximately 25 kDa), each amino-terminal portion of each chain comprising a variable region of approximately 100 to approximately 130 or more amino acids, and each carboxyl-terminal portion of each chain comprising a constant region (see 30 Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Kuby (1997) Immunology, Third Edition, WH Freeman and Company, New York). Here, the specific molecular antigen includes the target BTN1A1, which may be a BTN1A1 polypeptide, a BTN1A1 fragment, or a BTN1A1 epitope. The antibodies provided in this article include, but are not limited to, monoclonal antibodies, synthetic antibodies, recombinant antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, camel-derived antibodies, chimeric antibodies, intrabodies, and anti-idiotype (anti-Id) antibodies.

[0121] As used herein, unless otherwise stated, the term "monoclonal antibody" refers to an antibody that is the product of a single-cell clone or hybridoma, or a population of cells derived from a single cell. Monoclonal antibody is also intended to refer to antibodies produced by recombinant methods from immunoglobulin genes encoding both heavy and light chains, resulting in a single molecule of a particular type of immunoglobulin. The amino acid sequences of antibodies within monoclonal antibody products are substantially homogeneous, and the binding activity of antibodies within such products substantially exhibits the same antigen-binding activity. In contrast, polyclonal antibodies are derived from different B cells within a population; they are combinations of immunoglobulin molecules that bind to specific antigens. Each immunoglobulin in a polyclonal antibody can bind to different epitopes of the same antigen. Methods for producing both monoclonal and polyclonal antibodies are well known in the art (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) and Borrebaeck (ed.), Antibody Engineering: A Practical Guide, WH Freeman and Co., Publishers, New York, pp. 103-120 (1991)).

[0122] As used herein, unless otherwise stated, the term "human antibody" refers to an antibody having a human variable region and / or a human constant region, or a portion thereof corresponding to a human germline immunoglobulin sequence. Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 describes such human germline immunoglobulin sequences. Herein, human antibodies may include antibodies that bind to BTN1A1 and are encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence.

[0123] As used herein, unless otherwise stated, the term "chimeric antibody" refers to an antibody whose heavy and / or light chains are identical to or derived from corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or derived from corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al.). Proc. Natl. Acad. Sci. USA , 81: 6851-6855 (1984)).

[0124] As used herein, unless otherwise stated, the term "humanized antibody" refers to a chimeric antibody comprising a human immunoglobulin (e.g., an acceptor antibody), wherein the native complementarity-determining region ("CDR") residues are replaced by residues of the corresponding CDR of a non-human species (e.g., a donor antibody) having the desired specificity, affinity, and capability, such as mice, rats, rabbits, or non-human primates. In some cases, one or more FR region residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may have residues not present in the acceptor or donor antibody. These modifications are made to further enhance antibody performance. The heavy or light chain of the humanized antibody may substantially have all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin, and all or substantially all of the FRs are FRs of the human immunoglobulin sequence. The humanized antibody may have at least a portion of the immunoglobulin constant region (Fc), generally the constant region of a human immunoglobulin. For further details, see Jones et al. Nature , 321: 522-525 (1986); Riechmann et al., Nature , 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol . , 2: 593-596 (1992); Carter et al, Proc. Natl. Acd. Sci. USA 89: 4285-4189 (1992); and U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213 and 6,054,297.

[0125] As used herein, unless otherwise stated, the term “recombinant antibody” means an antibody prepared, expressed, created, or isolated by a recombinant manner. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant library of combined antibodies, antibodies isolated from transgenic and / or transchromosomally transgenic animals (e.g., mice or cows) of human immunoglobulin genes (see, for example, Taylor, LD et al., Nucl. AcidsRes.20: 6287-6295 (1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing of immunoglobulin gene sequences with other DNA sequences. Such recombinant antibodies may have variable and constant regions, including those derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The recombinant antibody may also undergo in vitro mutagenesis (or, when the animal is transgenic with a human Ig sequence, in vivo somatic mutagenesis), so that when derived from or related to human germline VH and VL sequences, the amino acid sequences of the VH and VL regions of the recombinant antibody may be sequences from a full set of human antibody germlines that are not naturally present in vivo.

[0126] As used herein, unless otherwise stated, a "neutralizing molecule" refers to a molecule that blocks the binding of BTN1A1 to its natural ligands such as GAL-1, GAL-9, NRP-2, or BTLA, and inhibits BTN1A1-mediated signaling pathways and / or other physiological activities. In some embodiments, the neutralizing molecule is a neutralizing antibody. In some embodiments, the neutralizing molecule comprises an antigen-binding fragment that immune-specifically binds to BTN1A1 or a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA. The IC50 of a neutralizing molecule or neutralizing antibody refers to the concentration of said molecule or antibody required to neutralize 50% of BTN1A1 in a neutralization assay. In a neutralization assay, the IC50 of said neutralizing molecule or neutralizing antibody may be in the range of 0.01-10 μg / ml. In some embodiments, said neutralizing molecule or neutralizing antibody may immune-specifically bind to BTN1A1. In some embodiments, the neutralizing molecule or neutralizing antibody may bind to a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA.

[0127] As used herein, unless otherwise stated, the term "antigen-binding fragment" and similar terms refer to a portion of an antibody that comprises amino acid residues that bind specifically to an antigen and confer specificity and affinity to the antigen. An antigen-binding fragment may be referred to as a functional fragment of the antibody. Antigen-binding fragments can be monovalent, bivalent, or polyvalent.

[0128] Molecules containing antigen-binding fragments include, for example, Fd, Fv, Fab, F(ab'), F(ab)2, F(ab')2, single-chain Fv (scFv), diabody, triabody, tetrabody, minibody, or single-domain antibody. scFv can be monovalent or bivalent. Other molecules containing antigen-binding fragments include, for example, heavy-chain or light-chain polypeptides, variable-region polypeptides, or CDR polypeptides or portions thereof, provided that such antigen-binding fragments retain binding activity. Such antigen-binding fragments can be found, for example, in Harlow and Lane. Antibodies: A Laboratory Manua l Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCHPublisher, Inc.; Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol, 178: 497-515 (1989) and Day, ED, Advanced Immunochemistry , Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antigen-binding fragment may be a polypeptide having an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0129] The heavy chain of an antibody is a polypeptide chain of approximately 50-70 kDa, in which the amino-terminal portion includes a variable region of approximately 120-130 or more amino acids, and the carboxyl-terminal portion includes a constant region. Based on the amino acid sequence of the constant region of the heavy chain, the constant region can be one of five different types, called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ). The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while µ and ε contain approximately 550 amino acids. When bound to a light chain, these different types of heavy chains produce five well-known classes of antibodies: IgA, IgD, 30IgE, IgG, and IgM. IgG includes four subclasses: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0130] The light chain of an antibody refers to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion includes a variable region of approximately 100 to approximately 110 or more amino acids, and the carboxyl-terminal portion includes a constant region. The approximate length of the light chain is 211–217 amino acids. Based on the amino acid sequence of the constant domain, two different types exist, referred to as kappa (κ) and lambda (λ). The amino acid sequences of the light chain are well known in the art. The light chain can be a human light chain.

[0131] The variable domain or variable region (CDR) of an antibody refers to a portion of the antibody's light or heavy chain, typically located at the amino terminus of the light or heavy chain. It is approximately 120-130 amino acids long in the heavy chain and approximately 100-110 amino acids long in the light chain, and is responsible for the binding and specificity of each specific antibody to its specific antigen. The sequences of the CDRs vary considerably among different antibodies. Sequence differences are concentrated in the CDRs, while smaller variable portions within the CDRs are called frame regions (FRs). The CDRs of both the light and heavy chains are primarily responsible for the antibody-antigen interaction. The amino acid positions used in this paper are numbered according to the EU index, as in Kabat et al. (1991). Sequences of proteins of immunological interest As described in the 5th edition of (USDepartment of Health and Human Services, Washington, DC). Variable regions can be human variable regions.

[0132] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VHβ-sheet frame of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VLβ-sheet frame of an antibody. Therefore, CDR is a variable region sequence scattered within the frame region sequence. CDR regions are well known to those skilled in the art, for example, as defined by Kabat as the most highly variable region within the variable (V) domain of an antibody (Kabat et al.). J. Biol. Chem . 252:6609-6616 (1977); Kabat, Adv. Prot. Chem . 32: 1-75 (1978)). The CDR region sequence has also been structurally defined by Chothia as those residues that are not part of the conserved β-sheet framework and are therefore capable of adapting to different conformations (Chothia and Lesk, J. Mol. Biol . 196: 901-917 (1987)). Both terms are well known in the art. The position of the CDR within the typical antibody variable domain has been determined through comparisons of many structures (Al-Lazikani et al., J. Mol. Biol 273: 927-948 (1997); Morea et al., Methods 20: 267-279 (2000)). Because the number of residues in the hypervariable region varies in different antibodies, other residues relative to the typical position are usually numbered a, b, c, etc., and these numbers are adjacent to the number of residues in the typical variable domain numbering scheme (Al-Lazikani et al., supra (1997). This naming convention is well known to those skilled in the art.

[0133] For example, CDRs defined according to standard naming are listed in Table 1 below.

[0134] Table 1: CDR Definition

[0135] One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an immunoadhesin. Immunoadhesins can be incorporated into CDRs as part of a larger polypeptide chain, or CDRs can be covalently linked to another polypeptide chain, or they can be non-covalently incorporated into CDRs. CDRs allow immunoadhesins to bind to specific antigens of interest.

[0136] "Frame" or "FR" residues refer to those variable domain residues located on the flanks of the CDR. FR residues are found in, for example, chimeric, humanized, human domain antibodies, biantibodies, linear antibodies, and bispecific antibodies. FR residues are variable region residues other than the hypervariable region residues defined herein.

[0137] As used herein, unless otherwise stated, the term "isolated" when referring to the use of an antibody means that the antibody is substantially free of cellular material or other contaminating proteins of cellular or tissue origin, and / or other contaminating components from which the antibody is derived, or substantially free of chemical precursors or other chemicals during chemical synthesis. The term "substantially free of cellular material" includes articles of antibodies in which the antibody is isolated from cellular components of cells, either isolated from or recombinantly generated from the cells. Thus, antibodies substantially free of cellular material include articles of antibodies having less than about 30%, 20%, 10%, or 5% (dry weight) of heterologous proteins (also referred to herein as "contaminating proteins"). In some embodiments, when the antibody is recombinantly generated, it is substantially free of culture medium, for example, the culture medium being less than about 20%, 10%, or 5% of the volume of the protein article. In some embodiments, when the antibody is generated by chemical synthesis, it is substantially free of chemical precursors or other chemicals, for example, it is isolated from chemical precursors or other chemicals involved in protein synthesis. Therefore, such antibody products contain less than about 30%, 20%, 10%, or 5% (by dry weight) of a chemical precursor or compound other than the target antibody. Contaminant components may also include, but are not limited to, materials that would interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other protein- or non-protein solutes. In some embodiments, the antibody will be purified to (1) according to the Lowry method (Lowry... et al.(J. Bio. Chem. 193: 265-275, 1921) The antibody must be greater than 95% by weight, for example 99% by weight, (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary cup sequencer, or (3) homogeneous under reducing or non-reducing conditions by using Coomassie Brilliant Blue or preferred silver yellow staining. Isolated antibodies include recombinant intracellular in situ antibodies, as at least one component of the antibody's native environment is absent. However, typically, isolated antibodies will be prepared by at least one purification step. In specific embodiments, the antibodies provided herein are isolated.

[0138] As used herein, unless otherwise stated, the terms “polynucleotide,” “nucleotide,” “nucleic acid,” “nucleic acid molecule,” and other similar terms, including DNA, RNA, mRNA, etc., are used interchangeably.

[0139] As used herein, unless otherwise stated, the term "isolated" for nucleic acid molecules refers to nucleic acid molecules that are isolated from other nucleic acid molecules present in natural sources. Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In certain embodiments, the nucleic acid molecules encoding antibodies provided herein are isolated or purified.

[0140] Unless otherwise stated, the terms "bind" or "binding" as used herein refer to the interaction between molecules. Interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The strength of the total non-covalent interactions between an antibody and a single epitope of a target molecule such as BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA is the antibody's affinity for that epitope. "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen).

[0141] The affinity of a molecule X (e.g., an antibody) for its binding partner Y can usually be determined by the dissociation constant (K). D ( ) indicates. Low-affinity antibodies typically bind slowly to antigens and dissociate easily, while high-affinity antibodies typically bind quickly to antigens and maintain binding for a longer time. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of this disclosure. "K D "or "K D The value can be measured by means of determinations known in the art, such as by combining determinations. KD It can be determined in radiolabeled antigen binding assays (RIA), for example, using the Fab type of the antibody of interest and its antigen (Chen et al., (1999)). J. Mol. Biol . 293: 865-881). K D or K D The value can also be measured using surface plasmon resonance of Biacore, for example using Biacore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ), or using biolayer interferometry, such as the Octet QK384 system (Fortebio, MenloPark, CA).

[0142] As used herein, unless otherwise stated, a molecule is considered to be capable of “immunospecific binding” to a second molecule if such binding exhibits specificity and affinity of the antibody for its associated antigen. If such binding involves an antigen recognition site of the antibody, the antibody immune-specifically binds to the target region or conformation (“epitope”) of the antigen. If other antigens have a certain sequence or conformational similarity recognized by the antigen recognition site, an antibody that immune-specifically binds to a particular antigen may bind to the other antigen with lower affinity, as determined by, for example, immunoassay, BIACORE® assay, or other assays known in the art. Antibodies typically do not bind to completely unrelated antigens. Some antibodies (and their antigen-binding fragments) do not cross-react with other antigens. Antibodies can also bind to other molecules in a non-immunospecific manner, such as binding to FcR receptors, relying on binding domains in other regions / domains of the antibody that do not involve antigen recognition sites, such as the Fc region.

[0143] An antibody or antigen-binding fragment that specifically binds to an antigen or epitope containing a glycosylation site can bind to the antigen or epitope in either a glycosylated or non-glycosylated form. In some embodiments, the antibody or antigen-binding fragment preferentially binds to a glycosylated antigen or epitope relative to a non-glycosylated antigen or epitope. This preferential binding can be determined by binding affinity. For example, an antibody or antigen-binding fragment that preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1 can have a lower Kc than that exhibited by the relatively non-glycosylated BTN1A1. D K D Binding to glycosylated BTN1A1. In some embodiments, the antibody or antigen-binding fragment exhibits a K value smaller than that of relatively non-glycosylated BTN1A1. D Half of K DBinding to glycosylated BTN1A1. In some embodiments, the antibody or antigen-binding fragment exhibits a K value smaller than that of relatively non-glycosylated BTN1A1. D At least 10 times K D Binding to glycosylated BTN1A1. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A1. D To interact with the K of non-glycosylated BTN1A1 D Approximately 75%, approximately 50%, approximately 25%, approximately 10%, approximately 5%, approximately 2.5%, or approximately 1%.

[0144] An antibody or antigen-binding fragment that specifically binds to BTN1A1 can bind to a BTN1A1 monomer or a BTN1A1 dimer. In some embodiments, the antibody or antigen-binding fragment preferentially binds to a BTN1A1 dimer relative to a BTN1A1 monomer. BTN1A1 binding can occur, for example, with BTN1A1 expressed on the cell surface or with soluble BTN1A1 domain constructs, such as BTN1A1 extracellular domain (ECD) constructs (e.g., flag-tagged BTN1A1-ECD or BTN1A1-CED-FC fusion constructs). In some embodiments, the BTN1A1 monomer or dimer is glycosylated at one or more sites. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of the BTN1A1 dimer. D Compared to K bound to BTN1A1 monomer D Half the size. In some implementations, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer. D K, relative to BTN1A1 monomer binding D At least 10 times smaller. In some implementations, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer. D K, which binds to ABTN1A1 monomer D Approximately 75%, approximately 50%, approximately 25%, approximately 10%, approximately 5%, approximately 2.5%, or approximately 1%.

[0145] Preferred binding can also be determined by binding assays and indicated, for example, by mean fluorescence intensity (“MFI”). For example, an antibody or antigen-binding fragment that preferentially binds to glycosylated BTN1A1 can bind glycosylated BTN1A1 with an MFI higher than that exhibited by non-glycosylated BTN1A1. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A1 is at least twice that of binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A1 is at least three times that of binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A1 is at least five times, at least ten times, at least fifteen times, or at least twenty times that of binding to non-glycosylated BTN1A1.

[0146] As used herein, unless otherwise stated, a molecule referred to as “immunospecifically masking” the glycosylation of an antigen or epitope, or a designated glycosylation site thereof, means that it has the ability to (1) block the glycosylation site of a non-glycosylated antigen or epitope, thereby preventing the antibody or epitope from being glycosylated, or (2) bind to the glycosylated antigen or epitope, or bind at a designated glycosylation site of the glycosylated antigen or epitope, and prevent the physiological effects of the glycosylation, such as downstream signal transduction mediated by the glycosylation. For example, an antibody or antigen-binding fragment that immunospecifically masks the glycosylation of BTN1A1 means that the antibody or antigen-binding fragment (1) blocks the glycosylation site of a non-glycosylated BTN1A1 and prevents its glycosylation, or (2) binds to the glycosylated BTN1A1 and prevents the physiological effects of the glycosylation, such as immunosuppression mediated by the glycosylation. Another example is that the antibody or antigen-binding fragment that immune-specifically masks BTN1A1 glycosylation at N55 and N215 means that the antibody or antigen-binding fragment (1) blocks non-glycosylated BTN1A1 at N55 and N215 and prevents glycosylation at N55 and N215, or (2) binds glycosylated BTN1A1 at N55 and N215 and prevents the physiological effects of said glycosylation, such as immunosuppression mediated by said glycosylation.

[0147] As used herein, unless otherwise stated, the term "vehicle" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, stabilizer, or carrier administered in conjunction with a therapeutic agent. A "pharmaceutically acceptable carrier" is a carrier that is non-toxic to cells or mammals exposed to it at the dose and concentration used; it can be a sterile liquid, such as water and oil, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. As used herein, unless otherwise stated, the term "vehicle" refers to a substance used to introduce nucleic acid molecules into host cells. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, exosomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, said vectors may include one or more selectable marker genes and appropriate expression control sequences. For example, the selectable marker genes that may be included may provide antibiotic or toxin resistance, compensate for nutritional deficiencies, or provide critical nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well-known in the art. When two or more nucleic acid molecules (e.g., antibody heavy and light chains) are to be co-expressed, both nucleic acid molecules may be inserted, for example, into a single expression vector or different expression vectors. For single-vector expression, the encoding nucleic acid may be operatively ligated to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of nucleic acid molecules into host cells can be confirmed using methods well-known in the art. These methods include, for example, nucleic acid analysis, such as Northern blotting or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting of gene product expression, or other suitable analytical methods to test the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art will understand that the amount of nucleic acid molecule expressed is sufficient to produce the desired product (e.g., the anti-BTN1A1 antibody provided by the present invention), and will further understand that the expression level can be optimized using methods well-known in the art to obtain sufficient expression.

[0148] As used herein, unless otherwise stated, the term "host cell" refers to a specific target cell transfected with a nucleic acid molecule and its progeny or potential progeny. The progeny of such cells may differ from the parent cells transfected with the nucleic acid molecule due to mutations that may occur in the next generation, environmental influences, or the integration of the nucleic acid molecule into the host cell genome.

[0149] Unless otherwise stated, the term "subject" as used herein means an animal used as a subject of treatment, observation, and / or experimentation. "Animal" includes vertebrates and invertebrates such as fish, shellfish, reptiles, birds, and especially mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates such as monkeys, chimpanzees, apes, and humans.

[0150] As used herein, unless otherwise stated, the terms “cancer” or “cancerous” refer to a physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, blood cancers and solid tumors.

[0151] As used herein, unless otherwise stated, the term “treatment” when used in relation to a cancer patient means the effect of reducing the severity of cancer or delaying or slowing the development of cancer, including (a) inhibiting the growth of cancer or delaying the development of cancer, and (b) causing the regression of cancer, or delaying or minimizing one or more symptoms associated with the presence of cancer.

[0152] As used herein, unless otherwise stated, the terms “resistant” or “refractory” refer to the presence of residual cancer cells (e.g., lung cancer or breast cancer cells) in a patient’s tissue or organ (e.g., lung or breast cancer cells) even after intensive treatment.

[0153] When referring to therapeutic use, the term "responsiveness" or "response" refers to the degree of effectiveness of the treatment in alleviating or reducing symptoms of a disease being treated, such as anti-PD1 therapy or anti-PD-L1 therapy-resistant or refractory cancer. For example, when referring to the treatment of cells or objects, the term "enhanced responsiveness" refers to an improvement in effectiveness in alleviating or reducing symptoms of a disease compared to a reference treatment (e.g., the same cell or object, or a different cell or object), when measured using any method known in the art. In some embodiments, the improvement in effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

[0154] As used herein, the terms “effective subject response,” “effective patient response,” and “effective patient tumor response” refer to any improvement in the therapeutic benefit for the patient. An “effective patient tumor response” can be, for example, a reduction of approximately 5%, approximately 10%, approximately 25%, approximately 50%, or approximately 100% in the rate of tumor progression. An “effective patient tumor response” can be, for example, a reduction of approximately 5%, approximately 10%, approximately 25%, approximately 50%, or approximately 100% in the physical symptoms of cancer. An “effective patient tumor response” can also be, for example, an improvement of approximately 5%, approximately 25%, approximately 50%, approximately 100%, approximately 200%, or higher in the patient’s response as measured by any suitable means, such as gene expression, cell count, analytical results, tumor size, etc.

[0155] Improvement in cancer or cancer-related disease can be characterized as a complete response or a partial response. A “complete response” is defined as the absence of clinically detectable disease, as measured using earlier abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF) measurements, or abnormal monoclonal protein assays. A “partial response” is defined as a reduction of at least approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, or approximately 90% in measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured volume of the tumor mass, or the number of abnormal monoclonal proteins) in the absence of new lesions. The term “treatment” anticipates both complete and partial responses.

[0156] As used herein, the term "probability" generally refers to an increased probability of an event. When used to refer to the effectiveness of a patient's tumor response, the term "probability" generally anticipates an increased probability that the rate of tumor progression or tumor cell growth will decrease. When used to refer to the effectiveness of a patient's tumor response, the term "probability" can also refer to indicators that demonstrate improved tumor progression, such as increased mRNA or protein expression.

[0157] The term "prediction" generally refers to something determined or informed in advance. When used to "predict" the effectiveness of cancer treatment, for example, the term "prediction" can mean that the likelihood of a cancer treatment outcome can be determined at the outset, before treatment begins or before the treatment cycle has substantially progressed.

[0158] As used herein, the term "monitoring" generally refers to the observation, supervision, regulation, surveillance, tracking, or monitoring of activity. For example, the term "monitoring the effectiveness of a compound" refers to tracking the effectiveness of cancer treatment in patients or in tumor cell cultures. Similarly, when used individually or in connection with patient compliance in clinical trials, the term "monitoring" refers to tracking or confirming that a patient is actually taking the tested drug as prescribed. For example, monitoring can be performed by tracking the expression of mRNA or protein biomarkers.

[0159] As used herein, the term "tumor" refers to all vegetative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. As used herein, "vegetative" refers to any form of abnormally regulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Therefore, "vegetative cells" include both malignant and benign cells exhibiting abnormally regulated or unregulated cell growth.

[0160] As used herein, unless otherwise stated, the term "therapeutic effective amount" refers to the quantity of an agent (e.g., an antibody described herein or any other agent described herein) sufficient to reduce and / or improve the severity and / or duration of a given disease, disorder, or condition and / or associated symptoms. A therapeutic effective amount of an agent including a therapeutic agent may be (1) necessary to reduce or improve the progression or development of a given disease, disorder, or condition, (ii) necessary to reduce or improve the recurrence, development, or onset of a given disease, disorder, or condition, and / or (iii) necessary to improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy different from the administration of the antibody provided herein). Therapeutic effective amounts of substances / molecules / agents disclosed herein (e.g., anti-BTN1A1 antibody) may vary depending on factors such as an individual's disease condition, age, sex, and weight, and the ability of the substance / molecule / agent to elicit a desired response in the individual. A therapeutic effective amount encompasses a quantity in which any toxicity or adverse effect of the substance / molecule / agent does not exceed the therapeutically beneficial effect.

[0161] As used herein, unless otherwise stated, the term "application" means the act of injecting or physically delivering a substance present outside the body into a patient, for example, through mucous membranes, intradermis, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. When a disease, disorder, or condition, or its symptoms, is being treated, the application of a substance is generally performed after the onset of the disease, disorder, or condition, or its symptoms. When a disease, disorder, or condition, or its symptoms, is being prevented, the application of a substance is generally performed before the onset of the disease, disorder, or condition, or its symptoms.

[0162] A “biological marker” or “biomarker” is a substance whose detection indicates a specific biological state, such as the presence of cancer. In some embodiments, a biomarker may be measured individually. In other embodiments, several biomarkers may be measured simultaneously. In some embodiments of the methods provided herein, BTN1A1 is a biomarker indicating the presence of cancer. In some embodiments, PD-L1 is a biomarker indicating the presence of cancer. In some embodiments, BTN1A1 and PD-L1 may be used in combination to indicate the presence of cancer (e.g., cancer responsive to treatment with, for example, an anti-BTN1A1 antibody, or cancer resistant or refractory to anti-PD1 or anti-PD-L1 therapy).

[0163] In some implementations, a “biomarker” indicates an alteration in mRNA expression levels that may be associated with disease risk or progression, or with susceptibility to a given treatment. In some implementations, the biomarker is a nucleic acid, such as mRNA or cDNA (e.g., BTN1A1 or PD-L1 mRNA or cDNA).

[0164] In another embodiment, a "biomarker" refers to an alteration in the expression level of a peptide or protein that may be associated with the risk or progression of disease, or a patient's susceptibility to treatment. In some embodiments, the biomarker may be a peptide or protein, or a fragment thereof (e.g., BTN1A1 or PD-L1 protein). The relative level of a specific protein can be determined by methods known in the art. For example, antibody-based methods, such as Western blotting, enzyme-linked immunosorbent assay (ELISA), or other methods may be used.

[0165] As used herein, the term "expressed" or "expressed" means an RNA nucleic acid molecule transcribed from a gene, said RNA nucleic acid molecule being at least partially complementary to one of the two nucleic acid strands of said gene. The term "expressed" or "expressed" as used herein also means a protein, polypeptide, or a portion thereof translated from an RNA molecule.

[0166] The term "level" refers to the amount, accumulation, or rate of a biomarker molecule (e.g., BTN1A1 or PD-L1). Levels can be expressed, for example, by the amount or rate of synthesis of gene-encoded messenger RNA (mRNA), gene-encoded polypeptides or proteins, or the amount or rate of synthesis of biomolecules accumulating in a cell or biological fluid. The term "level" also refers to the absolute or relative amount of molecules in a sample measured under steady-state or non-steady-state conditions.

[0167] As used herein, the terms “determine,” “measure,” “evaluate,” “assess,” and “analyze” generally refer to any form of measurement and include determining the presence of an element. These terms include quantitative and / or qualitative determinations. Assessments can be relative or absolute. Assessing presence can include determining the quantity of something present, as well as determining whether it exists or not.

[0168] As used herein, the term "sample" generally refers to a material or mixture of materials that contains one or more components of interest in liquid form.

[0169] As used herein, "biological sample" refers to a sample obtained from a biological object, including samples of biological tissue or fluid origin obtained or collected in vivo or in situ. Biological samples also include samples from portions of biological objects containing precancerous or cancerous cells or tissues. These samples can be, but are not limited to, organs, tissues, and cells isolated from mammals. Exemplary biological samples include, but are not limited to, cell lysates, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organelles, biological fluids, blood samples, urine samples, skin samples, etc. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, etc.

[0170] 5.2 Molecules possessing antigen-binding fragments that specifically bind to BTN1A1 or BTN1A1 ligands. This document provides molecules having an antigen-binding fragment that immune-specifically binds to BTN1A1 or a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, thereby enabling the molecule to inhibit the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the molecule can inhibit the formation of BTN1A1-BTN1A1 ligand complexes (e.g., complexes comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1A1). In some embodiments, the molecule can disrupt the formation of BTN1A1-BTN1A1 ligand complexes (e.g., complexes comprising GAL-251, GAL-9, NRP-2, or BTLA and BTN1A1). In some embodiments, the molecule is an antibody, including anti-BTN1A1 antibody, anti-GAL-1 antibody, anti-GAL-9 antibody, anti-NRP-2 antibody, or anti-BTLA antibody. In some embodiments, an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) binds to a fragment or epitope of BTN1A1 or a BTN1A1 ligand. In some embodiments, the antigen-binding fragment specifically binds to a BTN1A1 dimer. In some embodiments, the BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA epitope may be a linear epitope. In some embodiments, the BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA epitope may be a conformational epitope. In some embodiments, the BTN1A1 epitope is found in the BTN1A1 dimer but not in the BTN1A1 monomer. In some embodiments, the molecular inhibition of BTN1A1 or BTN1A1-BTN1A1 ligand complexes by antigen-binding fragments that specifically bind to BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA, as provided herein, is immunosuppressive.

[0171] In some embodiments, the molecule is an anti-glycosylated BTN1A1 antibody or an antigen-binding fragment comprising an anti-glycosylated BTN1A1 antibody. In some embodiments, the molecule is an anti-BTN1A1 dimer antibody or an antigen-binding fragment comprising an anti-BTN1A1 dimer antibody. In some embodiments, the molecule is a molecule having an antigen-binding fragment that specifically binds to BTN1A1, as described, for example, in International Patent Application PCT / US20106 / 064436 (filed December 1, 2016), U.S. Provisional Application 62 / 513389 (filed May 21, 2017), or U.S. Provisional Application No. 62 / 513393 (filed May 21, 2017), the entire contents of which are incorporated herein by reference. In some embodiments, the molecule comprises an antigen-binding fragment or VH, VL, or CDR sequence of an anti-BTN1A1 antibody STC703, STC810, STC820, STC1011, STC1012, or STC1029 or a humanized variant thereof, as described in U.S. International Patent Application PCT / US20106 / 064436, U.S. Provisional Application No. 62 / 513389, or U.S. Provisional Application No. 62 / 513393.

[0172] In some embodiments, the molecule is not STC810 ​​as described in International Patent Application No. PCT / US20126 / 64436, or its antigen-binding fragment or VH, VL or CDR amino acid sequence.

[0173] In some embodiments, the molecule is an anti-GAL-1 antibody or includes an anti-GAL-1 antibody. Antigen-binding fragment. In some embodiments, the molecule is an anti-GAL-1 antibody or an antigen-binding fragment comprising an anti-GAL-1 antibody as described in International Patent Application PCT / US1974 / 047783 (Publication No. WO20 / 013388A3), the entire contents of which are incorporated herein by reference.

[0174] In some embodiments, the molecule is an anti-GAL-9 antibody or an antigen-binding fragment comprising an anti-GAL-9 antibody. In some embodiments, the molecule is an anti-GAL-9 antibody or an antigen-binding fragment comprising an anti-GAL-9 antibody as described in International Patent Application PCT / FR20 / 051498 (e.g., disclosed as WO20125 / 185875A2), the entire contents of which are incorporated herein by reference.

[0175] In some embodiments, the molecule is an anti-NRP-2 antibody or an antigen-binding fragment comprising an anti-NRP-2 antibody. In some embodiments, the molecule is an anti-NRP-2 antibody or an antigen-binding fragment comprising an anti-NRP-2 antibody as described in International Patent Application PCT / US2002 / 069179 (e.g., disclosed as WO2002 / 143665A1), the entire contents of which are incorporated herein by reference.

[0176] In some embodiments, the molecule is an anti-NRP-2 antibody or an antigen-binding fragment comprising an anti-NRP-2 antibody. In some embodiments, the molecule is, for example, an anti-NRP-2 antibody or an antigen-binding fragment comprising said anti-NRP-2 antibody as described in International Patent Application No. PCT / US2002 / 069179 (e.g., disclosed as WO2002 / 143665A1) or PCT / US2007069185 (e.g., disclosed as WO2002 / 143666A2), the entire contents of which are incorporated herein by reference.

[0177] In some embodiments, the molecule is an anti-BTLA antibody or an antigen-binding fragment comprising an anti-BTLA antibody. In some embodiments, the molecule is an anti-BTLA antibody or an antigen-binding fragment comprising an anti-BTLA antibody as described in International Patent Applications PCT / US20126 / 64385 (filed December 21, 1976), PCT / US2010 / 043182 (e.g., disclosed as WO2011 / 014438, etc.), PCT / EP2010 / 053356 (e.g., disclosed as WO2010 / 106051A1), and PCT / US2002 / 084792 (e.g., disclosed as WO1988 / 076560A2), the entire contents of which are incorporated herein by reference.

[0178] In some embodiments, the molecule is an anti-BTN1A1 antibody or an antigen-binding fragment comprising an anti-BTN1A1 antibody. In some embodiments, the molecule is an anti-BTN1A1 antibody or an antigen-binding fragment comprising an anti-BTN1A1 antibody as described in International Patent Application PCT / US20106 / 064436 (filed December 21, 1976), the entire contents of which are incorporated herein by reference.

[0179] On one hand, this document provides a molecule comprising an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of a BTN1A1 ligand, such as galactolectin-1 (GAL-1), galactolectin-9 (GAL-9), NRP-2 (Nrp-2), or B- and T-lymphocyte attenuating proteins (BTLA). In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-1. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-9. In some embodiments, the molecule inhibits the binding of BTN1A1 to NRP-2. In some embodiments, the molecule inhibits the binding of BTN1A1 to BTLA. In some embodiments, the molecule completely inhibits the binding of a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA. In some embodiments, the molecule can at least partially inhibit the binding of a BTN1A1 ligand to BTN1A1, such as Gal-1, Gal-9, NRP-2, or BTLA. In some embodiments, the molecule can inhibit the binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1A1 ligand such as GAL-1, GAL-9, NRP-2, or BTLA to BTN1A1. In some embodiments, the binding of BTN1A1 to a BTN1A1 ligand, or its inhibition, is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule can inhibit the binding of BTN1A1 ligands to BTN1A1, such as GAL-1, Gal-9, NRP-2, or BTLA, with IC50 values ​​less than 1 μm, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some implementations, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

[0180] In some embodiments, the molecule includes an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of two or more BN1A1 ligands to BTN1A1, such as GAL-1, GAL-9, NRP-2, or BTLA. In some embodiments, the molecule can inhibit the binding of GAL-1 and GAL-9 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of NRP-2 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A1.

[0181] In some embodiments, the molecule includes an antigen-binding domain that binds to the extracellular domain (ECD) of BTN1A1.

[0182] On the other hand, a molecule provided herein includes an antigen-binding fragment that immune-specifically binds to a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the antigen-binding fragment immune-specifically binds to GAL-1, and the molecule inhibits the binding of GAL-1 to BTN1A1. In some embodiments, the antigen-binding fragment immune-specifically binds to GAL-9, and the molecule inhibits the binding of GAL-9 to BTN1A1. In some embodiments, the antigen-binding fragment immune-specifically binds to NRP-2, and the molecule inhibits the binding of NRP-2 to BTN1A1. In some embodiments, the antigen-binding fragment immune-specifically binds to BTLA, and the molecule inhibits the binding of BTLA to BTN1A1. In some embodiments, the molecule can completely inhibit the binding of the BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1A1. In some embodiments, the molecule can at least partially inhibit the binding of a BTN1A1 ligand to BTN1A1, such as GAL-1, GAL-9, NRP-2, or BTLA. In some embodiments, the molecule can inhibit the binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1A1 ligand such as GAL-1, GAL-9, NRP-2, or BTLA to BTN1A1. In some embodiments, the binding of BTN1A1 to a BTN1A1 ligand, or its inhibition, is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule can inhibit the binding of BTN1A1 ligands to BTN1A1, such as GAL-1, Gal-9, NRP-2, or BTLA, with IC50 values ​​less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.In some embodiments, the neutralization assay is a surface plasmon resonance assay, a biolayer interference assay, a co-immunoprecipitation assay, a FRET or TR-FRET assay, or an ELISA.

[0183] In some embodiments, the antigen-binding fragment of the molecule provided herein can bind to BTN1A1 ligands such as GAL-1, Gal-9, NRP-2, or BTLA with dissociation constants of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the antigen-binding fragment may bind to GAL-1 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the antigen-binding fragment may bind to Gal-9 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 μM or less. In some embodiments, the antigen-binding fragment may be bound to NRP-2 with a binding dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.In some embodiments, the antigen-binding fragment may bind to BTLA with a binding dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.

[0184] In some embodiments, the molecule can modulate the activity or signal transduction of BTN1A1, or modulate the activity or signal transduction of a complex of BTN1A1 and a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA).

[0185] In some embodiments, the molecule can regulate T cell activity. In some embodiments, the T cells are CD8+ cells. In some embodiments, the molecule can increase T cell activation or T cell proliferation. In some embodiments, the molecule can inhibit T cell apoptosis.

[0186] N-glycosylation is a post-translational modification that begins in the endoplasmic reticulum (ER) and is subsequently processed in the Golgi apparatus (Schwarz and Aebi, Curr. Opin. Struc. Bio . , 21(5): 576-582 (2011)). This type of modification is first catalyzed by a membrane-associated oligosaccharide transferase (OST) complex that transfers a pre-formed glycan composed of oligosaccharides to an asparagine (Asn) side chain acceptor located within the NXT motif (-Asn-X-Ser / Thr-) (Cheang and Reithmeier, Methods (Helenius and Aebi, 41: 451-459, 2007). Science , 291(5512): 2364-9(2001). The addition or removal of sugars from preformed glycans is mediated by a group of glycotransferases and glycosidases that tightly regulate the N-glycosylation cascade in a cell- and location-dependent manner.

[0187] In some embodiments, the molecule has an antigen-binding fragment that selectively binds to one or more glycosylation motifs of BTN1A1. In some embodiments, the antigen-binding fragment immune-specifically binds to glycopeptides having glycosylation motifs and adjacent peptides. In some embodiments, the antigen-binding fragment immune-specifically binds to peptide sequences located in three dimensions near one or more glycosylation motifs. In some embodiments, the antigen-binding fragment selectively binds to one or more glycosylation motifs of a BTN1A1 dimer, relative to one or more glycosylation motifs of a BTN1A1 monomer.

[0188] In some embodiments, the antigen-binding fragment binds to glycosylated BTN1A1 (e.g., glycosylated BTN1A1 dimer), relative to K binding to non-glycosylated BTN1A1. D Its KD is less than 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the antigen-binding fragment binds to glycosylated BTN1A1, relative to the KD bound to non-glycosylated BTN1A1. D , its K D Less than 50%. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to K binding to non-glycosylated BTN1A1 D Small 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%. In some embodiments, the antigen-binding fragment binds to K-type glycosylated BTN1A1. D Compared to K binding to non-glycosylated BTN1A1 D At least 10 times smaller.

[0189] Specific glycosylation sites of a particular BTN1A1 isotype or variant may differ from those of the specific BTN1A1 isotype or variant at amino acid positions 55, 215, or 449. In these cases, those skilled in the art can determine the glycosylation sites corresponding to N55, N215, and N449 of the human BTN1A1 exemplified above, based on sequence alignment and other common knowledge in the art. Similarly, molecules having antigen-binding fragments are also provided herein that immunely and specifically bind to the glycosylated form of the BTN1A1 isotype or variant relative to the non-glycosylated BTN1A1 isotype or variant. The glycosylation sites of the BTN1A1 isotype or variant may be the corresponding sites of N55, N215, and N449 of the human BTN1A1 sequence provided above.

[0190] In some embodiments, the molecule has an antigen-binding fragment that specifically binds to glycosylated BTN1A1 (e.g., a glycosylated BTN1A1 dimer). In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55, N215, and / or N449. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at position N55. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at position N215. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at position N449. In some embodiments, the antigen-binding fragment binds specifically to one or more glycosylated motifs. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55 and N215. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N215 and N449. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55 and N449. In other embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55, N215, and N449.

[0191] In some embodiments, the molecule has an antigen-binding fragment that specifically binds to glycosylated BTN1A1, whereby the antigen-binding fragment preferentially binds to glycosylated BTN1A1 (e.g., glycosylated BTN1A1 dimers) relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55, N215, and / or N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at position N55 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at position N215 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at position N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to one or more glycosylation motifs. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55 and N215 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N215 and N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55 and N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55, N215, and N449 relative to non-glycosylated BTN1A1.

[0192] Preferred binding can be determined by binding affinity. For example, antibodies or antigen-binding fragments that preferentially bind to glycosylated BTN1A (e.g., glycosylated BTN1A dimers) can exhibit a Ka ratio relative to non-glycosylated BTN1A. D Smaller K D Binding to glycosylated BTN1A. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Compared to K binding to non-glycosylated BTN1A D Half the size. In some embodiments, the antibody or antigen-binding fragment binds to the K group of glycosylated BTN1A. D Compared to the non-glycosylated BTN1A-bound K D At least twice as small. In some embodiments, the antibody or antigen-binding fragment binds to glycosylated BTN1A, K D Compared to the K exhibited by non-glycosylated BTN1A DAt least 5 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to glycosylated BTN1A, K D Compared to the K exhibited by non-glycosylated BTN1A D At least 10 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Compared to the K of non-glycosylated BTN1A D At least 15 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Compared to the K of non-glycosylated BTN1A D At least 20 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Compared to K binding to non-glycosylated BTN1A D At least 25 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Compared to the K exhibited when bound to non-glycosylated BTN1A D At least 30 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Compared to K binding to non-glycosylated BTN1A D At least 40 times smaller. In some embodiments, the antibody or antigen-binding fragment is bound to K+ that binds to glycosylated BTN1A. D Compared to K binding to non-glycosylated BTN1A D At least 50 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D 75%. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D 50%. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D 25%. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D 10%. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D5%. In some embodiments, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D 2.5%. In some implementations, the antibody or antigen-binding fragment binds to the K+ of glycosylated BTN1A. D Approximately K binding to non-glycosylated BTN1A D 1%.

[0193] Preferred binding can also be determined by binding assays, such as fluorescence intensity (“MFI”). For example, an antibody or antigen-binding fragment that preferentially binds to glycosylated BTN1A1 (e.g., a glycosylated BTN1A dimer) exhibits a higher MFI when binding to glycosylated BTN1A compared to when binding to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least twice as high as the MFI of non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least twice as high as the MFI of non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least three times as high as the MFI of non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least five times as high as the MFI of non-glycosylated BTN1A. In some embodiments, the MFI (Mean Function Index) of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least 10 times that of binding to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least 15 times that of binding to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least 20 times that of binding to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least 25 times that of binding to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to glycosylated BTN1A is at least 30 times that of binding to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment bound to glycosylated BTN1A is at least 40 times that of the MFI bound to non-glycosylated BTN1A. In some embodiments, the MFI of the antibody or antigen-binding fragment bound to glycosylated BTN1A is at least 50 times that of the MFI bound to non-glycosylated BTN1A.

[0194] In some embodiments, the antigen-binding fragment immunospecifically masks BTN1A glycosylation (i.e., glycosylated BTN1A dimers at positions N55, N215, and / or N449). In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylation at position N55 of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylation at position N215 of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylation at position N449 of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks one or more glycosylation motifs of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylations at positions N55 and N215 of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylations at positions N215 and N449 of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylations at positions N55 and N449 of BTN1A. In some embodiments, the antigen-binding fragment immunospecifically masks the glycosylations at positions N55, N215, and N449 of BTN1A.

[0195] In some embodiments, the molecule has an antigen-binding fragment that selectively binds to the BTN1A1 dimer relative to the BTN1A1 monomer. In some embodiments, the BTN1A1 dimer is expressed on the cell surface. In some embodiments, the BTN1A1 dimer is a soluble protein fragment of BTN1A1, such as an extracellular domain construct of BTN1A1, such as an Fc-fusion protein construct (e.g., BTN1A1-ECD-Fc). In some embodiments, the BTN1A monomer is an extracellular domain construct of BTN1A1, such as a flag-tagged or His6-tagged BTN1A1-ECD construct. In some embodiments, the molecule that selectively binds the BTN1A1 dimer is a molecule that selectively binds glycosylated BTN1A1 as provided herein. In some embodiments, the preferential binding of the BTN1A1 monomer to the BTN1A1 dimer is determined by using, for example, surface plasmon resonance analysis (e.g., Biacore) to determine the preferential binding of the BTN1A1-ECD-His6 or BTN1A1-ECD-Flag construct to the BTN1A1-ECD-Fc construct.

[0196] In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) DThe amount is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the antigen-binding fragment binds to the K+ of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D Less than 50%. In some embodiments, the antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D Small 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 10 times smaller.

[0197] Preferred binding can be determined by binding affinity. For example, antibodies or antigen-binding fragments that preferentially bind to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) bind to the K-terminal of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The KD is smaller relative to that of binding BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the KD of the antibody or antigen-binding fragment binding to a BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer) is smaller. D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D Half the size. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least twice as small. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 5 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). DK, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 10 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 15 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 20 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 25 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 30 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 40 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D K, relative to the binding of BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 50 times smaller. In some embodiments, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer). D Approximately K relative to the bound BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer). D 75%. In some embodiments, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer). D Approximately K relative to the bound BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer). D50%. In some embodiments, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer). D Approximately K relative to the bound BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer). D 25%. In some embodiments, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer). D Approximately K relative to the bound BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer). D 10%. In some embodiments, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer). D Approximately K relative to the bound BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer). D 5%. In some embodiments, the antibody or antigen-binding fragment binds to the K of the BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer). D Approximately K relative to the bound BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer). D 1%.

[0198] Preferred binding can also be determined by binding assays, such as fluorescence intensity (“MFI”). For example, an antibody or antigen-binding fragment that preferentially binds to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) exhibits a higher MFI when binding to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer) than when binding to a BTN1A1 monomer. In some embodiments, the MFI of the antibody or antigen-binding fragment binding to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least twice that of binding to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment binding to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least three times that of binding to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the median free fraction (MFI) of the antibody or antigen-binding fragment bound to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least 5 times higher than the MFI exhibited when bound to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment bound to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least 10 times higher than the MFI exhibited when bound to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment bound to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least 15 times higher than the MFI exhibited when bound to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment binding to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least 20 times higher than the MFI exhibited when binding to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment binding to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least 25 times higher than the MFI exhibited when binding to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment binding to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) is at least 30 times higher than the MFI exhibited when binding to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the MFI of the antibody or antigen-binding fragment bound to the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer) is at least 40 times that of the MFI exhibited upon binding to the BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer).In some embodiments, the M of the antibody or antigen-binding fragment binding to the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer) is at least 50 times greater than the MFI exhibited when binding to the BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer).

[0199] In some embodiments, the antibody or antigen-binding fragment preferentially binds to the glycosylated dimer BTN1A1 relative to the glycosylated monomer BTN1A1. The two BTN1A1 monomers in the glycosylated BTN1A1 dimer may be independently glycosylated at the same or different positions. In some embodiments, one monomer in the BTN1A1 dimer is not glycosylated. The glycosylated BTN1A1 monomers in the glycosylated BTN1A1 dimer may be glycosylated at positions N55, N215, and / or N449. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at position N55. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at position N215. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at position N449. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at both positions N55 and N215. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at positions N55 and N449. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at positions N215 and N449. In some embodiments, the glycosylated BTN1A1 monomer is glycosylated at positions N55, N215, and N449.

[0200] 5.2.1 Antibodies and other molecules with antigen-binding fragments In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can be IgG, IgM, IgA, IgD, or IgE. The anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can also be a chimeric antibody, an affinity-matured antibody, a humanized antibody, or a human antibody. The anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can also be a camel-derived antibody, an intracellular antibody, or an anti-idiotype (anti-Id) antibody. In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can be a polyclonal antibody or a monoclonal antibody.

[0201] Antibodies can be produced from any animal source, including birds and mammals. In some embodiments, antibodies are from sheep, mice (e.g., mice and rats), rabbits, goats, guinea pigs, camels, horses, or chickens. Additionally, newer technologies allow for the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows for the production of specific antibodies without animal immunization, as described in U.S. Patent No. 6,946,546, which is incorporated herein by reference in its entirety. These technologies are further described in Marks (1992); Stemmer (1994); Gram et al. (1992); Barbas et al. (1994); and Schier et al. (1996), which are incorporated herein by reference in their entirety.

[0202] Methods for producing polyclonal antibodies in various animal species, and methods for producing various types of monoclonal antibodies, including humanized, chimeric, and fully humanized antibodies, are well known in the art. For example, the following U.S. patents provide descriptions of implementations of such methods, which are incorporated herein by reference: U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797; 4,472,509; 4,606,855; 4,703,003; 4,742,15 9; 4,767,720; 4,816,567; 4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066; 5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,6 98; 5,627,052; 5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657; 5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365 ,157; ​​6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259; 6,861,572; 6,875,434; 6,891,024; 7,407,659; and 8,178,098, the full text of which is incorporated herein by reference.

[0203] Molecules containing antigen-binding fragments that specifically bind to BTN1A1 or specifically bind to BTN1A1 ligands (such as GAL-1, Gal-9, NRP-2, BTLA) (including anti-BTN1A1 antibodies or anti-BTN1A1 ligand antibodies (e.g., GAL-1, GAL-9, NRP-2, or BTLA)) can also be produced by any method known in the art for producing peptides, such as in vitro synthesis, recombinant DNA production, etc. Humanized antibodies can be produced using recombinant DNA technology. The antibodies described herein can also be produced using recombinant immunoglobulin expression technology. The recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Patent Nos. 4,816,397 (Boss et al.), U.S. Patent Nos. 6,331,415 and 4,816,567 (all granted to Cabilly et al.), British Patent GB 2,188,638 (Winter et al.), and British Patent GB 2,209,757, the entire contents of which are incorporated herein by reference. Recombinant expression techniques for immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al., GeneExpression Technology Methods in Enzymology Vol. 185. AcademicPress The findings were found in (1991) and Borreback, Antibody Engineering, WH Freeman (1992); the full text of which is incorporated herein by reference. Further information on the generation, design, and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, . Academic Press , San Diego (1993).

[0204] In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody is a human antibody. Human antibodies can be prepared by a variety of methods known in the art, including the aforementioned phage display method using an antibody library derived from human immunoglobulin sequences (see U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publications Nos. WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741). Human antibodies can be produced using transgenic mice that do not express functional endogenous immunoglobulins but do express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or through homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional by introducing human immunoglobulin gene loci, separately or simultaneously, through homologous recombination. Specifically, homozygous deletion of the JH region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then cultured to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized using conventional methods with selected antigens, such as all or part of a BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA polypeptide, or glycosylated BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA polypeptide, or a BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA polypeptide dimer. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques (see, for example, U.S. Patent No. 5,916,771). The human immunoglobulin transgenes carried by the transgenic mice undergo rearrangement during B cell differentiation, followed by species switching and somatic mutations. Therefore, using such techniques, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For an overview of this technique for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol(See 13:65-93, the entire text of which is incorporated herein by reference). For a detailed discussion of the techniques for producing human antibodies and human monoclonal antibodies and the protocols for producing such antibodies, see, for example, International Publications WO98 / 24893, WO96 / 34096 and WO96 / 33735; and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598, the entire text of which is incorporated herein by reference. Additionally, companies such as Abgenix, Inc. (Freemont, Calif) and MEDAREX (Princeton, NJ) may use similar techniques to those described above to provide human antibodies against selected antigens.

[0205] In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody is a chimeric antibody, such as an antibody having an antigen-binding sequence from a non-human donor, said sequence being grafted onto a heterologous non-human, human, or humanized sequence (e.g., frame and / or constant domain sequences). In one embodiment, the non-human donor is a rat. In one embodiment, the antigen-binding sequence is synthetic, for example, obtained by mutagenesis (e.g., phage display screening of a human phage library, etc.). In one embodiment, the chimeric antibody may have a mouse V region and a human C region. In one embodiment, the mouse light chain V region is fused with a human κ light chain. In one embodiment, the mouse heavy chain V region is fused with a human IgG1C region.

[0206] Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985. Science 229:1202; Oi et al., 1986, Biotechniques 4:214; Gillies et al., 1989, J. Immunol. Methods US Patents 125:191-202; and US Patents 6,311,415, 5,807,715, 4,816,567 and 4,816,397; the entire contents of all these patents are incorporated herein by reference. Chimeric antibodies comprising one or more CDRs from non-human species and framework regions from human immunoglobulin molecules can be produced using a variety of techniques known in the art, including, for example, CDR transplantation (EP239400; International Publication No. WO91 / 09967; and US Patents 5,225,539, 5,530,101 and 5,585,089), bonding or surface reconstruction (EP592,106; EP519,596; Padlan, 1991, ...). Molecular Immunology 28(4 / 5): 489-498; Studnicka et al., 1994, Protein Engineering 7:805; and Roguska et al., 1994, Proc. Natl. Acad. Sci USA91:969) and Chain Reorganization (US Patent 5,565,332). The entire contents of all these patents are incorporated herein by reference.

[0207] An exemplary process for producing recombinant chimeric anti-BTN1A1 or anti-BTN1A1 ligands (e.g., GAL-1, Gal-9, NRP-2, or BTLA) antibodies may include the following: a) Constructing an expression vector encoding and expressing an antibody heavy chain using conventional molecular biology methods, wherein the CDRs and variable regions of a mouse anti-BTN1A1 or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby generating a vector for expressing a chimeric antibody heavy chain; b) Constructing an expression vector encoding and expressing an antibody light chain of a mouse anti-BTN1A1 or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) monoclonal antibody using conventional molecular biology methods, thereby generating a vector for expressing a chimeric antibody light chain; c) Transferring the expression vector to host cells using conventional molecular biology methods to generate transfected host cells for expressing chimeric antibodies; and d) Culturing transfected cells using conventional cell culture techniques to generate chimeric antibodies.

[0208] An exemplary process for producing recombinant humanized anti-BTN1A1 antibodies may include the following steps: a) constructing an expression vector encoding and expressing the antibody heavy chain using conventional molecular biology methods, wherein a minimal portion of the CDR and variable region framework required for donor antibody binding specificity is derived from a non-human immunoglobulin, such as a murine anti-BTN1A1 or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) monoclonal antibody, the remainder of which is derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody heavy chain; b) constructing an expression vector encoding and expressing the antibody light chain using conventional molecular biology methods, wherein a minimal portion of the CDR and variable region framework required for donor antibody binding specificity is derived from a non-human immunoglobulin, such as a murine anti-BTN1A1 or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), the remaining portion of which is derived from a human immunoglobulin, thereby producing a vector for expressing the humanized antibody heavy chain; The minimum portion of the CDR and variable region framework required for specific antibody binding is derived from non-human immunoglobulins, such as mouse anti-BTN1A1 or anti-BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) monoclonal antibodies, while the remainder of the antibody is derived from human immunoglobulins, thereby producing a vector expressing the humanized antibody light chain; c) transferring the expression vector into host cells using conventional molecular biology methods to produce transfected host cells for expressing humanized antibodies; and d) culturing transfected cells using conventional cell culture techniques to produce humanized antibodies.

[0209] For any exemplary method, host cells can be co-transfected with an expression vector that may contain different optional markers, but is preferably identical except for the heavy and light chain coding sequences. This method provides equivalent expression of the heavy and light chain peptides. Alternatively, a single vector encoding both heavy and light chain peptides can be used. The coding sequences for the heavy and light chains may include cDNA or genomic DNA or both. The host cells used to express the recombinant antibody can be bacterial cells such as *Escherichia coli*, or more preferably eukaryotic cells (such as Chinese hamster ovary (CHO) cells or HEK-293 cells). The choice of expression vector depends on the choice of host cell and can be selected to give it the desired expression and regulatory characteristics in the selected host cell. Other cell lines that can be used include, but are not limited to, CHO-K1, NSO, and PER. C6 (Crucell, Leiden, Netherlands). Furthermore, codon usage can be optimized when the selected host cell requires species-specific codon usage bias and enhances protein expression. For example, for CHO cell expression, the DNA encoding the antibody can be incorporated into *Armillaria globulus* (…). Cricetulus griseus (The Chinese hamster ovary cells are derived from the bacteria described above) preferentially used codons. Codon optimization methods can be used to promote the desired increased expression in host cells (see, for example, Wohlgemuth, I. et al.). Philos.Trans.R.Soc.Lond.BBiol. Sci. 366 (1580): 2979-2986 (2011); Jestin, JL et al. J. Mol. Evol. 69(5):452-457(2009); Bollenbach, T. et al., GenomeRes. 17(4): 401-404(2007); Kurland, CG, et al. Prog. Nucleic Acid Res. Mol. Biol. 31: 191-219 (1984); Grosjean, H et al., Gene 18(3): 199-209(1982)).

[0210] In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody may be a monoclonal antibody. In some embodiments, the anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody may be a polyclonal antibody. Animals may be inoculated with an antigen, such as a BTN1A1 peptide or glycosylated BTN1A1 peptide, BTN1A1 dimer peptide, GAL-1 peptide, GAL-9 peptide, NRP-2 peptide, or BTLA peptide, to generate antibodies specific to the BTN1A1 peptide or glycosylated BTN1A1 peptide, BTN1A1 dimer, GAL-1 peptide, GAL-9 peptide, NRP-2 peptide, or BTLA peptide. Typically, the antigen binds to or conjugates with another molecule to enhance the immune response. The conjugate may be any peptide, polypeptide, protein, or non-protein substance that binds to the antigen used to elicit an immune response in the animal. Antibodies produced in animals in response to antigen inoculation include a variety of different molecules (polyclonal antibodies) produced by B lymphocytes from multiple individual antibody production processes. When given the correct conditions for polyclonal antibody production in an animal, most antibodies in the animal's serum recognize aggregate epitopes on the antigenic compound on which the animal has been immunized.

[0211] This specificity can be further enhanced by affinity purification, selecting only those antibodies that recognize the antigen or epitope of interest. Methods for producing monoclonal antibodies (MAbs) can begin along the same route as the preparation of polyclonal antibodies. In some embodiments, rodents such as mice and rats are used to produce monoclonal antibodies. In some embodiments, rabbit, sheep, or frog cells are used to produce monoclonal antibodies. The use of rats is well-known and offers certain advantages. Mice (e.g., BALB / c mice) are routinely used and generally yield a higher percentage of stable fusions.

[0212] Hybridoma technology involves fusing a single B lymphocyte from a mouse pre-immunized with a BTN1A1 peptide, glycosylated BTN1A1 peptide, BTN1A1 dimer peptide, GAL-1 peptide, GAL-9 peptide, NRP-2 peptide, or BTLA peptide with immortalized myeloma cells (typically mouse myeloma). This technology provides a method for unlimited passage and propagation of monoclonal antibody-producing cells, thereby generating an unlimited quantity of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0213] In one embodiment, the antibody is derived from the immunoglobulin monovariable domain of a camel antibody, preferably from the heavy chain of a camel antibody lacking a light chain, and is referred to as V. HH domain sequences, or Nanobodies™. Nanobodies™ (Nb) are minimal functional fragments or single variable domains (V) of naturally occurring single-chain antibodies. H H), and is known to those skilled in the art. They are derived from antibodies that only contain heavy chains, as seen in camels (Hamers-Casterman et al., Nature , 363(6428): 446-8(1993); Desmyter et al, NatStructBiol , 3(9): 803-11. (1996)). Immunoglobulins without light polypeptide chains were discovered in the "camel" family. "Camel" includes Old World camels (Bactrian camels (Camelus b actrianus) and Dromedary camels (Camelus dromedarius)) and New World camels (e.g., alpacas (Lamapaccos), llamas (Lama glama), guanicoes (Lama guanicoe), and veal (Lama vicugna)). Single-chain variable domain heavy chain antibodies are defined as Nanobody™ or V H H antibodies. The small size and unique biophysical properties of Nbs are superior to conventional antibody fragments in recognizing uncommon or hidden epitopes and binding to cavities or active sites of protein targets. Furthermore, Nbs can be engineered as multispecific and multivalent antibodies, linked to reporter molecules, or humanized. Nbs are stable, survive in the gastrointestinal system, and are readily fabricated.

[0214] By integrating two antigen-binding sites with different specificities into a single construct, bispecific antibodies possess the ability to bind two discrete antigens with fine specificity, thus holding great potential as therapeutic agents. Bispecific antibodies can be prepared by fusing two hybridomas, each capable of producing different immunoglobulins. Bispecific antibodies can also be generated by linking two scFv antibody fragments while omitting the Fc portion present in whole immunoglobulins. Each scFv unit in these constructs can consist of a variable domain from each of the heavy (VH) and light (VL) antibody chains, interconnected by synthetic peptide linkers, which are typically genetically engineered to minimize immunogenicity while maintaining maximum resistance to proteolysis. Individual scFv units can be linked using various techniques, including incorporating short polypeptide spacers (typically less than 10 amino acids) bridging the two scFv units, thereby producing bispecific single-chain antibodies. Therefore, the resulting bispecific single-chain antibody contains two pairs of VH / VL pairs with different specificities on a single polypeptide chain, thereby separating the VH and VL domains in the corresponding scFv units by sufficiently long polypeptide linkers to allow intramolecular association between the two domains, and the resulting scFv units are kept short enough to prevent unwanted association between the VH domain of one scFv unit and the VL domain of another scFv unit by polypeptide spacers that are adjacent to each other.

[0215] Examples of molecules having an antigen-binding fragment that specifically binds to BTN1A1, glycosylated BTN1A1, BTN1A1 dimer, GAL-1, GAL-9, NRP-2, or BTLA include, but are not limited to: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an “Fd” fragment consisting of VH and CH1 domains; (iii) an “Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) a “dAb” fragment consisting of a VH domain; (v) a separated CDR region; (vi) an F(ab')2 fragment comprising a bivalent fragment of two linked Fab fragments; (vii) a single-chain Fv molecule (“scFv”) wherein the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) a bispecific single-chain Fv dimer (see U.S. Patent No. 5,091, 513); and (ix) biantibodies, multivalent or multispecific fragments constructed by gene fusion (US Patent Application Publication 20050214860), Fv, scFv or biantibody molecules can be stabilized by binding disulfide bonds linking the VH and VL domains. Mini antibodies with scFv linked to the CH3 domain can also be prepared (Hu et al., Cancer Res., 56(13): 3055-61(1996)).

[0216] Antibody-like binding peptide mimics are also included in the implementation scheme. Murali et al., CellMol. Biol . , 49(2):209-216 (2003) describes “antibody-like binding peptide mimics” (ABiPs), which are peptides used as attenuated antibodies and have some advantages such as a longer serum half-life and a less cumbersome synthesis method. The full text of the paper is incorporated herein by reference.

[0217] Standard techniques known to those skilled in the art can be used to introduce mutations into nucleotide sequences encoding antigen-binding fragments or antibodies provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutations leading to amino acid substitutions. In some embodiments, the derivative comprises amino acid substitutions of fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substituents, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In a particular embodiment, the derivative is subjected to conserved amino acid substitutions at one or more predicted non-essential amino acid residues. “Conserved amino acid substitution” refers to an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be randomly introduced along all or part of the coding sequence, for example, through saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein is expressed, and the activity of said protein is determined.

[0218] In some embodiments, the molecules provided herein can be chemically modified, for example by covalently linking any type of molecule to an antibody. For example, but not limited to, antibody derivatives include chemically modified antibodies, such as those derived by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or linking to cellular ligands or other proteins. Any of these many chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, antibodies may contain one or more non-classical amino acids.

[0219] The molecules provided herein may have framework regions (e.g., human or non-human fragments) known to those skilled in the art. These framework regions may be, for example, naturally occurring or common framework regions. In a particular embodiment, the framework regions of the antibodies provided herein are human (see, for example, Chothia et al., 1998, J. Mol. Biol. 278: 457-479). See also Kabat et al. (1991), Sequences of proteins of immunological interest (Department of Health and Human Services, Washington, DC), 5th edition.

[0220] On the other hand, this document provides molecules having antigen-binding fragments that competitively block (e.g., in a dose-dependent manner) the BTN1A1 epitope of an anti-BTN1A1 antibody, the GAL-1 epitope of an anti-GAL-1 antibody, the anti-GAL-9 epitope of an anti-GAL-9 antibody, the anti-NRP-2 epitope of an anti-NRP-2 antibody, or the BTLA epitope of an anti-BTLA antibody. The molecule may be an antibody. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

[0221] In some embodiments, this document provides anti-BTLA antibodies that competitively block (e.g., in a dose-dependent manner) the BTN1A1 epitope described herein, the anti-GAL-1 epitope of an anti-GAL-1 antibody, the anti-GAL-9 epitope of an anti-GAL-9 antibody, the anti-NRP-2 epitope of an anti-NRP-2 antibody, or the anti-BTLA epitope of an anti-BTLA antibody.

[0222] In some embodiments, the molecules provided herein have high affinity for BTN1A1, glycosylated BTN1A1, BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers), GAL-1, GAL-9, NRP-2, BTLA, or peptides or fragments thereof, or epitopes thereof. In one embodiment, the molecules provided herein may be anti-BTN1A1 antibodies or anti-BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies that have higher affinity for BTN1A1 or said BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) than known antibodies (e.g., commercially available monoclonal antibodies discussed elsewhere herein). In certain embodiments, the molecules provided herein may be anti-BTN1A1 antibodies or anti-BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies that have an affinity for BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA antigens that is 2-10 times (or higher) higher than that of known anti-BTN1A1 antibodies or known anti-BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies, as determined by techniques described herein or techniques known to those skilled in the art (e.g., the Biacore assay). According to these embodiments, in one embodiment, the affinity of the antibody is assessed by a Biacore assay.

[0223] In some embodiments, the molecules provided herein may have an antigen-binding fragment that binds to BTN1A1, glycosylated BTN1A1, a BTN1A1 dimer (e.g., α-glycosylated BTN1A1 dimer), GAL-1, GAL-9, NRP-2, BTLA, or a polypeptide or polypeptide fragment or epitope, with a dissociation constant (K0). D The molecular weight is not greater than 1 μM, not greater than 100 nM, not greater than 10 nM, not greater than 1 nM, or not greater than 0.1 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 500 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 200 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, RP-2, or BTLA) antibody not exceeding 100 nM. In some embodiments, the molecule provided herein may be K. DAnti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 50 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, Gal-9, NRP-2, or BTLA) antibody not exceeding 20 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 10 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 5 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 2 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 1 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody, not exceeding 0.5 nM. In some embodiments, the molecule provided herein may be K. D Anti-BTN1A1 antibody or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody not exceeding 0.1 nM.

[0224] In some embodiments, the molecules provided herein can block or neutralize the activity of BTN1A1 or BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA). The molecules can be neutralizing antibodies. These neutralizing antibodies can block the binding of BTN1A1 to natural ligands such as GAL-1, GAL-9, NRP-2, or BTLA, and inhibit signaling pathways and / or other physiological activities mediated by BTN1A1 or BTN1A1-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) complexes. In neutralization assays, the IC50 of the neutralizing antibody can be in the range of 0.01-10 μg / ml. The IC50 of the neutralizing antibody may not be greater than 10 μg / ml. The IC50 of the neutralizing antibody may not be greater than 8 μg / ml. The IC50 of the neutralizing antibody may not be greater than 6 μg / ml. The IC50 of the neutralizing antibody may not be greater than 4 μg / ml. The IC50 of neutralizing antibodies may not exceed 2 μg / ml. The IC50 of neutralizing antibodies may not exceed 1 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.8 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.6 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.4 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.2 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.1 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.08 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.06 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.04 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.02 μg / ml. The IC50 of neutralizing antibodies may not exceed 0.01 μg / ml.

[0225] The molecules provided herein that have an antigen-binding fragment that specifically binds to BTN1A1 (e.g., glycosylated BTN1A1 or BTN1A1 dimer) or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) can be anti-BTN1A1 antibodies or anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies. The antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camel-derived antibodies, chimeric antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above antibodies. Non-limiting examples of functional fragments include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, disulfide-linked Fv (SdFv), Fd fragments, Fv fragments, biantibodies, triantibodies, tetraantibodies, and miniantibodies.

[0226] Specifically, the molecules described herein include immunoglobulin molecules and immunoactive portions of immunoglobulin molecules, such as molecules containing antigen-binding fragments that specifically bind to BTN1A1 (e.g., glycosylated BTN1A1 or BTN1A1 dimers) or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA). The immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0227] The molecules provided herein may be monospecific, bispecific, trispecific antibodies, or antibodies with higher multispecificity. Multispecific antibodies may be specific to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA), or may be specific to both BTN1A1 peptides or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) and heterotopes (e.g., heteropeptides or solid support materials). In certain embodiments, the antibodies provided herein are monospecific to a given epitope of a BTN1A1 peptide or BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) peptide and do not bind to other epitopes.

[0228] 5.2.2 Modifications and Derivatives Antigen-binding fragments with immune-specific binding to BTN1A1 (e.g., glycosylated BTN1A1 or BTN1A1 dimers) or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) can be further improved by screening for variants exhibiting the desired properties. For example, such improvement can be accomplished using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. In a particular embodiment, such phages can be used to display antigen-binding fragments, such as Fab and Fv or disulfide-stabilized Fv, expressed from a full library or a combined antibody library (e.g., human or mouse). Phages expressing antigen-binding fragments that bind to target antigens can be antigen-selected or identified, for example, using labeled antigens or antigens that bind to or capture on a solid surface or bead. Phages used for these methods are typically filamentous phages, including fd and ML3. The antigen-binding fragment is expressed as a protein recombinantly fused with phage gene III or gene VIII proteins. Examples of phage display methods that can be used to prepare antibodies or other molecules having the antigen-binding fragments described herein include Brinkman et al., J Immunol Methods, 182: 41-50 (1995); Ames et al. J. Immunol. Methods , 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol , 24: 952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al., Adv. Immunol. 57: 191-280 (1994); PCT Publications WO92 / 001047; WO90 / 02809; WO91 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; WO95 / 15982; WO95 / 20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658, Those disclosed in 727; 5733743 and 5969108; the entire contents of all of these are incorporated herein by reference.

[0229] As described in the references above, after phage selection, antibody-coding regions derived from phages can be isolated and used to generate whole antibodies, including humanized antibodies or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as detailed below, for example. For instance, techniques for recombinantly producing Fab, Fab', and F(ab')2 can also be employed using methods known in the art, such as PCT disclosure WO92 / 22324; Mullinax, RL, et al. BioTechniques , 12(6): 864-869 (1992); and Sawai et al., AM. J. Reprod. Immunol. 34: 26-34 (1995); Better, M et al. Science The entire contents of all these documents are incorporated herein by reference. Examples of techniques that can be used to produce single-chain Fv and antibodies include U.S. Patent Nos. 4,946,778 and 5,258,498; Huston, JS, et al. Methods in Enzymology 203: 46-88 (1991); Shu, L. et al., Proc. Natl. Acad. Sci. (USA)90: 7995-7999; and Skerra. A. et al., Science Those described in 240: 1038-1040 (1988); all of these contents are incorporated herein by reference.

[0230] Phage display technology can be used to increase the affinity of antibodies against BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or other molecules having an immune-specific binding affinity for BTN1A1 (e.g., glycosylated BTN1A1 or BTN1A1 dimer) or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) as described herein. This technology can be used to obtain high-affinity antibodies that can be used in the combinatorial methods described herein. This technology, called affinity maturation, uses mutagenesis or CDR walking and reselection of such receptors or ligands (or their extracellular domains) or their antigenic fragments to identify antibodies that bind to antigens with higher affinity than the initial antibody or parent antibody (see, e.g., Glasser, SM, etc.). J. Immunol.149: 3903-3913 (1992). Mutagenesis of the entire codon, rather than a single nucleotide, results in a semi-random complete set of amino acid mutations. Libraries can be constructed consisting of pools of variant clones, each clone differing by a single amino acid change in a single CDR, containing variants representing every possible amino acid substitution for each CDR residue. Mutants with enhanced binding affinity to antigens can be screened by contacting immobilized mutants with labeled antigens. Any screening method known in the art can be used to identify mutant antibodies with enhanced affinity to antigens (e.g., ELISA) (see, for example, Wu, H. et al.). Proc. Natl. Acad. Sci. (USA)95(11):6037-6042(1998); Yelton, DE et al., J. Immunol 155: 1994-2004 (1995). Alternatively, a CDR walk that randomizes the light chain can be used (see Schier et al., J. Mol. Biol. 263: 551-567 (1996)).

[0231] Random mutagenesis can be used in conjunction with phage display methods to identify modified CDRs and / or variable regions. Phage display technology can also be used to increase (or decrease) CDR affinity through directed mutagenesis (e.g., affinity maturation or “CDR-walking”). This technique uses a target antigen or a fragment thereof to identify antibodies with CDRs that have a higher (or lower) affinity for the antigen compared to the original or parental antibody (see, e.g., Glass, SM, etc.). J. Immunol. 149: 3903-3913 (1992)).

[0232] Methods for achieving this affinity maturity are described, for example, by Krause, JC, etc. MBio 2(1) Pii:e00345-10. doi: 10.1128 / mBio.00345-10(2011); Kuan, CT et al. Int. J. Cancer 10.1002 / ijc. 25645; Hackel, BJ, etc. J. Mol. Biol. 401(1): 84-96 (2010); Montgomery DL et al., MAbs1(5): 462-474 (2009); Gustchia, E. et al., Virology 393(1): 112-119 (2009); Finlay, WJ et al. J. Mol. Biol. 388(3): 541-558 (2009); Bostrom, J. et al. Methods Mol. Biol.525: 353-376 (2009); Steidl, S. et al. Mol. Immunol. 46(1): 135-144 (2008); and Barderas, R. et al. Proc. Natl. Acad. Sci. (USA)105(26): 9029-9034(2008); All of these contents This is cited here for reference.

[0233] This document also provides derivatives of any of the above-described molecules having an antigen-binding fragment that immune-specifically binds to BTN1A1 (e.g., glycosylated BTN1A1 or a BTN1A1 dimer) or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), which may be anti-BTN1A1 antibodies or BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies, but have one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications relative to the "parental" (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids can be glycosylated (e.g., altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-ethanolneuraminic acid, etc.), acetylated, PEGylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytically cleaved, or linked to cellular ligands or other proteins. In some embodiments, the altered carbohydrate modification regulates one or more of the following: antibody dissolution, promotion of subcellular antibody transport and secretion, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In some embodiments, the altered carbohydrate modification enhances antibody-mediated effector function compared to antibodies lacking carbohydrate modification. Carbohydrate modifications that lead to alterations in antibody-mediated effector function are well known in the art (e.g., see Shields, RL et al., J. Biol. Chem. 277(30): 26733-26740 (2002); Davies J et al., Biotechnology & Bioengineering, 74(4): 288-294 (2001); the entire contents of all these references are incorporated herein by reference). Methods for altering carbohydrate content are known to those skilled in the art, see, for example, Wallik, SC et al., J. Exp. Med. 168(3): 1099-1109 (1988); Tao, MH et al., J. Immunol. 143(8): 2595-2601 (1989); Routledge, EG et al., transplantation, 60(8): 847-53 (1995); Elliott, S. et al., Nature Biotechnol. 21: 414-21 (2003); Shields, RL et al., J. Biol. Chem. 277(30): 26733-26740 (2002); the entire contents of all these are incorporated herein by reference.

[0234] In some embodiments, the humanized antibody is a derived antibody. Such a humanized antibody comprises the substitution, deletion, or addition of one or more amino acid residues in a non-human CDR. Compared to a non-derived humanized antibody, a humanized antibody derivative may have substantially the same binding, better binding, or worse binding. In some embodiments, one, two, three, four, or five amino acid residues of the CDR are... Mutations, such as substitution, deletion, or addition.

[0235] The molecules and antibodies described herein can be modified chemically using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, and metabolic synthesis of tunicamycin. In one embodiment, the derivative molecule or derivative antibody has a function similar to or the same as the parent molecule or antibody. In another embodiment, the derivative molecule or derivative antibody exhibits altered activity against the parent molecule or parent antibody. For example, the derived antibody (or a fragment thereof) may bind more tightly to its epitope or be more resistant to proteolysis than the parent antibody.

[0236] Substitutions, additions, or deletions in derived antibodies can occur in the Fc region of the antibody, and thus can be used to alter the binding affinity of the antibody to one or more FcγRs. Methods for modifying antibodies that bind to one or more FcγRs are known in the art, see, for example, PCT disclosures WO04 / 029207, WO04 / 028564, WO99 / 58572, WO99 / 51642, WO98 / 23289, WO89 / 07142, WO88 / 07089 and U.S. Patent Nos. 5,843,597 and 5,642,821; the entire contents of all these patents are incorporated herein by reference. In some embodiments, the antibody or other molecule has an altered affinity for an activated FcγR, such as FcγRIIIA. Preferably, such modification also has an altered Fc-mediated effector function. Modifications affecting Fc-mediated effector function are well known in the art (see U.S. Patents 6,194,551 and WO00 / 42072). In some embodiments, modifications to the Fc region result in altered antibody-mediated effector function, altered binding to other Fc receptors (e.g., Fc-activated receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity (CDC) activity, altered phagocytic activity, or any combination thereof.

[0237] ADCC is a cell-mediated response in which antigen-nonspecific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies that bind to the surface of target cells, subsequently leading to target cell lysis (i.e., “killing”). The initial mediating cells are NK cells. NK cells express only FcγRIII, where FcγRIIIA is the activating receptor and FcγRIIIB is the repressive receptor; monocytes express FcγRI, FcγRII, and FcγRIII (Ravetch et al. (1991)). Annu. Rev. Immunol. (9: 457-92). ADCC activity can be expressed as the concentration of the antibody or Fc fusion protein at half-maximum lysis of target cells. Therefore, in some embodiments, where the lysis level is the same as the half-maximum lysis level of the wild-type control, the concentration of the antibody or Fc fusion protein of the present invention is at least 2, 3, 5, 10, 20, 50, or 100 times lower than the concentration of the wild-type control itself. Additionally, in some embodiments, the antibody or Fc fusion protein of the present invention may exhibit higher maximum target cell lysis compared to the wild-type control. For example, the maximum target cell lysis of the antibody or Fc fusion protein may be 10%, 15%, 20%, 25%, or more higher than that of the wild-type control.

[0238] The molecules and antibodies described herein can be modified to have enhanced potency. In some embodiments, the effector functions of the molecules and antibodies are modified, for example, to enhance ADCC and / or complement-dependent cytotoxicity (CDC). In some embodiments, these therapeutic molecules or antibodies have enhanced interactions with cytotoxic cells carrying Fc receptors. Enhancement of effector functions, such as ADCC, can be achieved by various methods, including introducing one or more amino acid substitutions into the Fc region. Furthermore, cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in said region. The homodimeric antibodies may also have improved internalization capacity and / or increased CDC and ADCC. (Caron et al., J. ExpMed., 176: 1191-95 (1992) and Shopes, BJ Immunol., 148: 2918-22 (1992).) Heterobifunctional cross-linking agents can also be used to prepare homodimeric antibodies with enhanced anticancer activity. Wolff et al., Cancer Research, 53: 2560-65 (1993). Additionally, antibodies or molecules with dual Fc regions can be modified to possess enhanced CDC and ADCC capabilities. Stevenson et al., Anti-Cancer Drug Design 3: 219-30 (1989).

[0239] The glycosylation pattern of the Fc region can also be engineered. Many antibody glycosylation forms have been reported to have a positive impact on effector functions, including ADCC. Therefore, engineering the carbohydrate components of the Fc region, particularly reducing core fucosylation, could also have enhanced therapeutic efficacy. (Shinkawa T. et al.) J Biol. Chem , 278: 3466-73 (2003); NiwaR, et al. Cancer Res. 64: 2127-33(2004); OkazakiA et al., J Mol. Chem. 336: 1239^19 (2004); and ShieldsRL et al., J Biol. Chem. 277: 26733-40 (2002). The antibodies or molecules with selected glycoforms described herein can be produced by a variety of means, including using glycosylation pathway inhibitors, mutant cell lines lacking or with reduced activity of specific enzymes in the glycosylation pathway, engineered cells with enhanced or knocked-out gene expression in the glycosylation pathway, and in vitro modification with glycosidases and glycosyltransferases. Methods for modifying the glycosylation of the Fc region and enhancing the therapeutic potency of antibodies or other molecules with antigen-binding fragments are known in the art. Rothman et al., Molecular Immunology 26:1113-1123 (1989); M.Mana et al. Nature Biotechnology 17: 176-180 (1999); Shields et al., JBC277: 26733-26740 (2002); Shinkawa et al., JBC278: 3466-3473 (2003); Bischoff et al., J. Biol. Chem. 265(26): 15599-15605 (1990); U.S. Patents 6,861,242 and 7,138,262 and U.S. Publication No. 2002 / 0124652; all the contents of which are incorporated herein by reference. It will be understood by those skilled in the art that the antibodies and molecules provided herein can be modified by any method known in the art to have enhanced therapeutic efficacy.

[0240] Derivative molecules or antibodies may also have altered half-lives (e.g., serum half-lives) in mammals, preferably humans. In some embodiments, such alterations result in half-lives greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. An increased half-life in humanized antibodies or other molecules in mammals, preferably humans, results in higher serum titers of said antibodies or other molecules in mammals, thus reducing the frequency of administration of said antibodies or other molecules and / or reducing the concentration of said antibodies or other molecules to be administered. Molecules or antibodies with increased in vivo half-lives can be produced using techniques known to those skilled in the art. For example, molecules or antibodies with increased in vivo half-lives can be produced by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor. The humanized antibodies described herein can be engineered to increase biological half-lives (see, for example, U.S. Patent No. 6,277,375). For example, the humanized antibodies described herein can be engineered in the Fc-hinge domain to have an increased in vivo or serum half-life.

[0241] By attaching polymer molecules such as high molecular weight polyethylene glycol (PEG) to the antibody or antibody fragment, molecules or antibodies with increased in vivo half-life as described herein can be generated. PEG can be linked to the molecule or antibody via site-specific coupling of PEG to the N- or C-terminus of the molecule or antibody, or via the epsilon-amino group present on a lysine residue, with or without a multifunctional linker. Linear or branched polymers that result in minimal loss of biological activity can be used for derivatization. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

[0242] The molecules or antibodies described herein can also be modified using the methods and conjugates described by Davis et al. (see U.S. Patent 4,179,337) to provide compositions that can be injected into the mammalian circulatory system without substantially any immunogenic reaction. Removing the Fc moiety reduces the likelihood of the antibody fragment causing an undesirable immune response; therefore, Fc-free antibodies can be used for prophylactic or therapeutic treatment. As mentioned above, antibodies can also be constructed as chimeric, partially or fully human to reduce or eliminate adverse immunological outcomes resulting from the administration of antibodies produced in other species or having sequences from other species to animals.

[0243] 5.2.3 Fusion Proteins and Conjugates This document provides molecules having immune-specific binding fragments to BTN1A1 (e.g., glycosylated BTN1A1 or BTN1A1 dimer) or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antigens, including anti-BTN1A1 antibodies and anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies. In some embodiments, such molecules are expressed as fusion proteins with other proteins or chemically conjugated to another moiety.

[0244] In some embodiments, the molecule is a fusion protein having an Fc moiety, wherein the Fc moiety can be modified by isotype or subtype alteration, can be chimeric or heterozygous, and / or can be modified, for example, to improve effector function, half-life control, tissue accessibility, enhance biophysical properties such as stability, and improve production efficiency (and at a lower cost). Many modifications and methods for constructing the disclosed fusion proteins are known in the art; see, for example, Mueller, JP, et al. Mol. Immun . 34(6):441-452(1997), Swann, PG, Curr. Opin. Immun. 20: 493-499 (2006), and Presta, LG, Curr. Opin. Immun 20: 460-470 (2008). In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a hybrid, such as a chimera having a constant IgG2 / IgG4 Fc region. Modifications to the Fc region include, but are not limited to, IgG4 being modified to prevent binding to Fc receptors and complement, IgG1 being modified to improve binding to one or more Fc receptors, IgG1 being modified to minimize effector function (amino acid alterations), IgG1 with altered / absent glycans (typically by altering the expression host), and IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region or less than the entire hinge region.

[0245] Another implementation includes IgG2-4 hybrids and IgG4 mutants, which have reduced binding to FcR, thus increasing their half-life. Representative IgG2-4 hybrids and IgG4 mutants are described in Angal et al. Molec. Immunol 30(1): 105-108 (1993); Muller et al. Mol. Immun34(6): 441-452 (1997); and U.S. Patent No. 6,982,323; the entire contents of all of these are incorporated herein by reference. In some embodiments, the IgG1 and / or IgG2 domains are deleted, for example, Angal et al. describe IgG1 and IgG2 with proline replacing serine at position 241.

[0246] In some embodiments, the molecule is a polypeptide having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.

[0247] In some embodiments, this document provides molecules having antigen-binding fragments that specifically bind to BTN1A1 (e.g., glycosylated BTN1A1 or a BTN1A1 dimer) or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA), which are linked, covalently bound, or form complexes with at least one moiety. Such a moiety may be, but is not limited to, a moiety that enhances the efficacy of the molecule as a diagnostic or therapeutic agent. In some embodiments, said moiety may be an imaging agent, a toxin, a therapeutic enzyme, an antibiotic, a radiolabeled nucleotide, etc.

[0248] The molecules described herein may include therapeutic portions (or one or more therapeutic portions). The molecules described herein may be antibodies that bind to or recombinantly fuse to therapeutic portions, such as cytotoxins, such as cell-inhibiting or cytotoxic agents, therapeutic agents, or radioactive metal ions, such as alpha emitters. Cytotoxins or cytotoxic agents include any agents that are harmful to cells. Therapeutic portions include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine); alkylating agents (e.g., diethyldichlorodiethylamine, thiotepa chlorambucil, milfalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP) and cisplatin); anthracene Cyclic antibiotics (e.g., daunorubicin (formerly daunorubicin) and doxorubicin); antibiotics (e.g., actinomycin D (formerly namycin), bleomycin, sclerotinib, and amiodarone (AMC)); auristatin molecules (e.g., auristatin PHE, auristatin F, monomethylauristatin E, bryostatin 1, and solatatin 10; see Woyke) et al., Antimicrob. AgentsChemother. 46: 3802-8 (2002), Woyke et al.,Antimicrob.Agents Chemother. 45: 3580-4(2001),Mohammad et al., Anticancer Drugs12: 735-40(2001),Wall et al., Biochem. Biophys. Res. Commun. 266: 76-80(1999),Mohammad et alInt. J. Oncol. 15: 367-72 (1999) All of these are incorporated herein by reference; hormones (e.g., glucocorticoids, progesterone, androgens, and estrogens), DNA repair enzyme inhibitors (e.g., etoposide or topotecan), kinase inhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al, Clin Cancer Res. 8(7): 2167-76 (2002); cytotoxic agents (e.g., paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, doxorubicin, daunomycin, dihydroxyanthraxetine, mitoxantrone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, natriuretic acid, and puromycin, and their analogues or homologs, and U.S. Patent Nos. 6,245,759, 6,399,633, and 6,383). The compounds disclosed in 790, 6,335,156, 6,271,242, 6,242,196, 6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, and 5,587,459)Farnesyltransferase inhibitors (e.g., Rl15777, BMS-214662 and, for example, U.S. Patents: 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501, 6,268,363, 6,265,4...) 22、6,248,756、6,239,140、6,232,338、6,228,865、6,228,856、6,225,322、6,218,406、6,211,193、6,187,786、6,169,096、6,159,984、6,143,766、6,133,303、6,127,366、6,124,465、6,124,295、6,103,723、6,093,737、6,090,948、6,080,870、6,077,853、6,071,935、6,066,738、6,0 Those disclosed in 63,930, 6,054,466, 6,051,582, 6,051,574 and 6,040,305); topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38; topotecan; 9-aminocamptothecin; GG-211 (GI147211); DX-8951f; IST-622; rubitecan; pyrazoline acridine; XR-5000; saintopin; UCE6; UCE1022; TAN-1518a; TAN-1518b; KT6006; KT6528; ED-110; NB-506; ED-110; BN-50). 6; and bulgarein; DNA minor groove binding agents, such as Hoescht dye 33342 and Hoechst dye 33258; zafyne; zanthoxyne; epiberberine; methoxyberberine; β-lapaquinone; BC-4-1; bisphosphonates (e.g., alendronate, incardronate, chlorotriphosphatase, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, pyrdronate, pamidronate, zoledronic acid);HMG-CoA reductase inhibitors (e.g., lovastatin, simvastatin, atorvastatin, pravastatin, fluvastatin, statins, cerivastatin, letrozole, atorvastatin, rosuvastatin, and atorvastatin); antisense oligonucleotides (e.g., those disclosed in U.S. Patent Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenine nucleoside deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); teimiramab (Zevalin®); tosiramabab (Bexxar®) and their pharmaceutically acceptable salts, solvates, cages, and prodrugs.

[0249] Furthermore, the molecules provided herein may be antibodies conjugated or recombinantly fused to a therapeutic or pharmaceutical moiety that modifies a given biological response. The therapeutic or pharmaceutical moiety should not be considered limited to standard chemotherapeutic agents. For example, the pharmaceutical moiety may be a protein, peptide, or polypeptide with the desired biological activity. Such proteins may include, for example, toxins such as abrin, castorin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, gamma-interferon, alpha-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, and apoptosis agents such as TNF-γ, AMII (see, for example, International Publication No. WO97 / 33899), AMIII (see, for example, International Publication No. WO97 / 34911), Fas ligand (see, for example, Jakahashi et al., 1994, J. Immunol., 6: 1567-1574), and VEGF (see, for example, International Publication No.). WO99 / 23105), anti-angiogenic agents, such as angiostatin, endostatin, or components of the coagulation pathway (e.g., tissue factor); or, biological response modifiers, such as lymphokines (e.g., interferon gamma, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-5 (“IL-5”), interleukin-6 (“IL-6”), interleukin-7 (“IL-7”), interleukin-9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12 (“IL-12”), interleukin-15 (… (“IL-15”), interleukin-23 (“IL-23”), granulocyte-macrophage colony-stimulating factor (“GM-CSF”) and granulocyte colony-stimulating factor (“G-CSF”), or growth factors (e.g., growth hormone (“GH”), or coagulants (e.g., calcium, vitamin K, tissue factors, such as but not limited to, Hagmann factor (factor XII), high molecular weight kininogen (HMWK), prokalopeptidase (PK), thrombin-factor II (prothrombin), factors V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipids and fibrin monomers).

[0250] Furthermore, the antibodies provided herein can be conjugated to therapeutic motifs such as radioactive metal ions, such as α-emitters, 213Bi, or macrocyclic chelators for conjugating radioactive metal ions to peptides, including but not limited to 131In, 131LU, 131Y, 131HO, and 131Sm. In some embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which can be linked to the antibody via a linker molecule. Such linker molecules are well known in the art and were described in Denardo et al., 1998. Clin Cancer Res 4(10): 2483-90; Peterson et al., 1999, Bioconjug. Chem 10(4): 553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8): 943-50, the full text of which is incorporated herein by reference.

[0251] The therapeutic portion or drug conjugated or recombined with an antibody that specifically binds to BTN1A1 (e.g., glycosylated BTN1A1 or a BTN1A1 dimer) or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) as described herein should be selected to achieve the desired preventive or therapeutic effect. In some embodiments, the antibody is a modified antibody. Clinicians or other medical personnel should consider the following factors when deciding which therapeutic portion or drug conjugated or recombined with the antibody provided herein: the nature of the disease, the severity of the disease, and the patient's condition.

[0252] In some embodiments, the portion may be an enzyme, hormone, cell surface receptor, toxin (e.g., abrin, ginseng A, Pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ginseng, gelonin, or pokeweed antiviral protein), protein (e.g., tumor necrosis factor, interferon (e.g., α-interferon, β-interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, or apoptosis agent (e.g., tumor necrosis factor-α, tumor necrosis factor-β)), or bioreactive modifier (e.g. Examples include lymphokines (e.g., interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”)), granulocyte-macrophage colony-stimulating factor (“GM-CSF”), granulocyte colony-stimulating factor (“G-CSF”) or macrophage colony-stimulating factor (“M-CSF”)), or growth factors (e.g., growth hormone (“GH”)), cytotoxins (e.g., cell-inhibiting or cytotoxic agents, such as paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide). Glycosides, teniposide, vincristine, vinblastine, colchicine, doxorubicin, doxorubicin, daunomycin, dihydroxyanthraxetine, mitoxantrone, scintillans, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, natriuretic acid, methylauristatin F (MMAF), methylauristatin E (MMAE; e.g., vedotin), and puromycins and their analogues or homologs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil). Pyrimidine diimide), alkylating agents (e.g., diethyldichlorodiethylamine, thiotepa chlorambucil, milfalan, BiCNU® (carmustine; BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., erythromycin (formerly donomycin) and doxorubicin), antibiotics (e.g., actinomycin (formerly namycin), bleomycin, scintillan and ampicillin (AMC), or antimitotic agents (e.g., vincristine and vinblastine).

[0253] The technique of conjugating such therapeutic components to antibodies is well known; see, for example, Amon et al. Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “ Antibodies For Drug Delivery ”, in CONTROLLED DRUG DELIVERY (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “ Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ”, in MONOCLONAL ANTIBODIES’84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. (eds.), 1985, pp. 475-506); “ Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ”, in MONOCLONAL ANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; Thorpe et al., Immunol. Rev. 62: 119-158(1982); Carter et al., Cancer J. 14(3): 154-169(2008); Alley et al., Curr. Opin. Chem. Biol . 14(4): 529-537(2010); Carter et al., Amer. Assoc. Cancer Res. Educ. Book. 2005(1): 147-154(2005); Carter et al., Cancer J. 14(3): 154-169(2008); Chari , Acc. Chem Res. 41(1): 98-107(2008); Doronina et al., Nat. Biotechnol. 21(7): 778-784(2003); Ducry et al., Bioconjug Chem. 21(1): 5-13(2010); Senter, Curr. Opin. Chem. Biol. 13(3): 235-244(2009); and Teicher, Curr Cancer Drug Targets. 9(8): 982-1004(2009).

[0254] In some embodiments, the molecules described herein may be conjugated with markers, such as peptides, to facilitate purification. In some embodiments, the markers are hexahistine peptides, hemagglutinin “HA” tags that correspond to epitopes derived from influenza hemagglutinin proteins (Wilson, IA et al., Cell, 37: 767-778 (1984)), or “FLAG” tags (Knappik, A et al., Biotechniques 17 (4): 754-761 (1994)).

[0255] In some embodiments, the portion may be an imaging reagent that can be detected in the analysis. Such an imaging reagent may be an enzyme, a cofactor, a radioactive label, a non-radioactive paramagnetic metal ion, a hapten, a fluorescent tag, a phosphorescent molecule, a chemiluminescent molecule, a chromophore, a light-emitting molecule, a bioluminescent molecule, a photoaffinity molecule, a colored particle, or a ligand such as biotin.

[0256] In some embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; the cofactor complex includes, but is not limited to, streptavidin / biotin and avidin / biotin; the fluorescent material includes, but is not limited to, umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; the luminescent material, for example, but not limited to, luminol; the bioluminescent material includes, but is not limited to, luciferase, luciferin, and jellyfish luminescent protein; the radioactive material includes, but is not limited to, bismuth (213Bi), carbon (14c), chromium (51Cr), cobalt (57CO), fluorine (18f), gadolinium (153Gd, 159Gd), gallium (68Ga, 67Ga), germanium (68Ge), holmium (166Ho), indium (115In, 113In, 1... 12In, 111In), Iodine (131I, 125I, 123I, 121I), Lanthanum (140La), Lutetium (177Lu), Manganese (54Mn), Molybdenum (99Mo), Palladium (103Pd), Phosphorus (32P), Praseodymium (142Pr), Promethium (149Pm), Rhenium (186Re, 188Re), Rhodium (105Rh), Ruthenium (97Ru), Samarium (153Sm) Scandium (47Sc), selenium (75Se), strontium (85Sr), sulfur (35S), technetium (99Tc), thallium (201Ti), tin (113Sn, 117Sn), tritium (3h), xenon (133Xe), ytterbium (169Yb, 175Yb), yttrium (90Y), zinc (65Zn); positron-emitting metals and non-radioactive paramagnetic metal ions obtained using various positron emission tomography methods.

[0257] Imaging agents can be conjugated directly or indirectly to molecules having antigen-binding fragments via intermediates (e.g., known linkers in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900, for metal ions that can be conjugated to antibodies and other molecules described herein for use as diagnostic agents. Some conjugation methods involve the use of metal chelate complexes, which are linked to antibodies using, for example, organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6α-diphenylglycyl-3. Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Fluorescein-labeled conjugates can be prepared in the presence of these conjugating agents or by reaction with isothiocyanates.

[0258] The molecules described herein can be conjugated with a second antibody to form antibody heteroconjugates as described by Segal in U.S. Patent 4,676,980. Such heteroconjugate antibodies may additionally bind to haptens (e.g., fluorescein), or cellular markers (e.g., 4-1-BB, B7-H4, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, ICOS, B7-H3, B7-H7, B7-H7CR, CD70, CD47) or cytokines (e.g., IL-7, IL-15, IL-12, IL-4, TGF-β, IL-10, IL-17, IFNY, Flt3, BLys) or chemokines (e.g., CCL21).

[0259] The molecules described herein can be attached to solid supports that can be used for immunoassays or purification of target antigens or other molecules capable of binding to target antigens, which are immobilized on the support by binding to antibodies or antigen-binding fragments described herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0260] This document also provides nucleic acid molecules (DNA or RNA) encoding any such antibodies, antigen-binding fragments, and antigen-binding fragments having immune-specific binding to BTN1A1 (e.g., glycosylated BTN1A1 or BTN1A1 dimers). Vector molecules (e.g., plasmids) capable of delivering or replicating said nucleic acid molecules are also provided. Nucleic acids can be single-stranded, double-stranded, and may contain both single-stranded and double-stranded portions.

[0261] Antibody-Drug Conjugates (ADCs) Because the molecules described herein can lead to the internalization of BTN1A1 into cells, this article also provides antibody-drug conjugates (ADCs) including any of the anti-BTN1A1 antibodies described herein.

[0262] In some implementations, this document provides antibody-drug conjugates, including antibody-drug conjugates of formulas (Ia) and (Ib):

[0263]

[0264] Or its pharmaceutically acceptable salt; in: A is a molecule containing an antigen-binding fragment; The two described cysteine ​​residues originate from an open cysteine-cysteine ​​disulfide bond in A; Each of X and X' is independently O, S, NH, or NR. 1 , where R 1 It is C 1-6 alkyl; W a =N-, =CH-, =CHCH2-, =C(R) 2 )-、or =CHCH(R 2 )-;W b -NH-、-N(R 1 )-, -CH2-, -CH2-NH-, -CH2-N(R 1 -CH2CH2- -CH(R) 2 - or -CH2CH(R) 2 )-; where R 1 and R 2 C is independent 1-6 alkyl; CTX is a cytotoxin; R can be any chemical group, or R may not exist. L 1 L 2 and L 3 Each of these is independently selected from the following connectors: -O-, -C(O)-, -S-, -S(O)-, -S(O)2-, -NH-, -NCH3-, -(CH2) q -, -NH(CH2)2NH-, -OC(O)-, -CO2-, -NHCH2CH2C(O)-, -C(O)NHCH2CH2NH-, -NHCH2C(O)-, -NHC(O)-, -C(O)NH-, -NCH3C(O)-, -C(O)NCH3-, -(CH2CH2O) p-(CH2CH2O) p CH2CH2-, -CH2CH2-(CH2CH2O) p -、-OCH(CH2O-)2、-(AA) r - Cyclopentyl, cyclohexyl, unsubstituted phenylene, and phenylene substituted with one or two of the following substituents: halogen, CF3-, CF3O-, CH3O-, -C(O)OH, -C(O)OC 1-3 Alkyl groups, -C(O)CH3, -CN, -NH-, -NH2, -O-, -OH, -NHCH3, -N(CH3)2, and C 1-3 alkyl; a, b, and c are each an independent integer of 0, 1, 2, or 3, provided that at least one of a, b, or c is 1; k and k’ Each of these is an integer that is either 0 or 1; Each p is an independent integer from 1 to 14; Each q is an independent integer from 1 to 12; Each AA is an amino acid; Each r is from 1 to 12; m is an integer from 1 to 4; n is an integer from 1 to 4; and A key represents a single key or a double key.

[0265] In some embodiments of the antibody-drug conjugate (ADC) of formula (Ib), R is selected from W, (L) as defined herein. 1 ) a 、(L 2 ) b 、(L 3 ) c Z, W-(L) 1 ) a -(L 2 ) b -(L 3 ) c 、(L 1 ) a -(L 2 ) b -(L 3 ) c -Z, and W-(L) 1 ) a -(L 2 ) b -(L 3 ) c -Z. In some implementations, R is selected from W, (L1 ) a 、(L 2 ) b 、(L 3 ) c and W-(L 1 ) a -(L 2 ) b -(L 3 ) c In some implementations, R is selected from Z, (L... 1 ) a -(L 2 ) b -(L 3 ) c -Z and W-(L) 1 ) a -(L 2 ) b -(L 3 ) c -Z.

[0266] In some embodiments of the antibody-drug conjugate (ADC) of formula (Ib), R is a detectable probe. In some embodiments, R is a fluorophore, chromophore, radiolabel, enzyme, ligand, antibody, or antibody fragment. In some embodiments, R is a ligand (e.g., a ligand specific to a receptor on tumor cells such as prostate-specific membrane antigen, or a ligand for virus-infected cells such as HIV-infected cells).

[0267] In some embodiments of the antibody-drug conjugate (ADC) of formula (Ib), R is obtained by amide, N-(C 1-6 Alkyl amides, carbamates, N-(C) 1-6 Alkyl) carbamates, amines, N-(C) 1-6 Alkylamines, ethers, thioethers, ureas, N-(C) 1-6 Alkyl)urea, or N,N-bis(C 1-6 Alkyl)urea bonds are attached to the rest of the connector molecule.

[0268] In certain embodiments of the antibody-drug conjugate (ADC) of formula (Ia) or (Ib), each L 1 L 2 and L 3 Independently selected from -NHC(O)-, -C(O)NH-, -(CH2CH2O) p -(CH2CH2O) p CH2CH2-, -CH2CH2-(CH2CH2O) p -、-OCH(CH2O-)2、-(AA) r- Unsubstituted phenyl groups and 1 or 2 groups selected from halogens, CF3-, CF3O-, CH3O-, -C(O)OH, -C(O)OC 1-3 Alkyl, -C(O)CH3, -CN, -NH-, -NH2, -O-, -OH, -NHCH3, -N(CH3)2 and C1-3 alkyl; wherein a, b and c are each independently 0 or 1; and each p and r is independently 1, 2 or 3. In some embodiments, L 1 L 2 and L 3 One or more are - (AA) r -, where -(AA) r It is ValCit (e.g., the first amino acid is valine, the second amino acid is citrulline, and r is 1). In some embodiments, L 1 L 2 and L 3 One or more of them are -(AA)r-, where -(AA)r- r It is ValAla (e.g., the first amino acid is valine, the second amino acid is alanine, and r is 1). In some embodiments, L 1 L 2 and L 3 One or more are -C(O)OH and -NH2-substituted phenylene groups. In some embodiments, L 1 L 2 and L 3 One or more are -C(O)O- and -NH-substituted phenylene oxides. In some embodiments, L 1 L 2 and L 3 One or more are -OC(O)- and -NH-substituted phenylene oxides. In some embodiments, L 1 L 2 and L 3 One or more are -O- and -NH-substituted phenylene groups. In some embodiments, L 1 L 2 and L 3 One or more of them are para-aminophenyl (PAB), which are optionally substituted with C(O)O-, -OC(O)-, or -O-. In some embodiments, L 1 It is -(CH2) q -, L 2 L does not exist. 3 It does not exist, and CTX is related to (L) 1 ) a -(L 2 ) b -(L 3 )c Linked via amide bonds. In some embodiments, L 1 It is -(CH2) q -, L 2 It is -(OCH2CH2) p -, L 3 It does not exist, and CTX is bonded to (L) via an amide bond. 1 ) a -(L 2 ) b -(L 3 ) c In some implementations, L 1 It is -(CH2CH2O) p -, L 2 It is -(CH2) q -, L3 is absent, and CTX is bonded to (L) via an amide bond. 1 ) a -(L 2 ) b -(L 3 ) c In some implementations, each L 1 Independently selected from -(CH2CH2O) p CH2CH2- and -CH2CH2-(CH2CH2O) p -, L 2 L does not exist. 3 It does not exist, and CTX is bonded to (L) via an amide bond. 1 ) a -(L 2 ) b -(L 3 ) c In some implementations, each L 1 Independently selected from -(CH2) q -、-(CH2CH2O) p -(CH2CH2O) p CH2CH2-, -CH2CH2-(CH2CH2O) p -、and-C(O)-,L 2 It is Val-Cit, L 3 It is PAB, and CTX is bonded to (L) via an amide bond. 1 ) a -(L 2 ) b -(L 3 ) c In some implementations, each L 1 Independently selected from -(CH2) q-、-(CH2CH2O) p -(CH2CH2O) p CH2CH2-, -CH2CH2-(CH2CH2O) p -、and-C(O)-,L 2 It is Val-Cit, L 3 It is PAB, and CTX is bonded to (L) via an amide bond. 1 ) a -(L 2 ) b -(L 3 ) c In some implementations, each L 1 Independently selected from -(CH2) q -、-(CH2CH2O) p -(CH2CH2O) p CH2CH2-, -CH2CH2-(CH2CH2O) p -、and-C(O)-,L 2 It is Val-Ala, L 3 It is PAB, and CTX is bonded to (L) via an amide bond. 1 ) a -(L 2 ) b -(L 3 ) c .

[0269] In some embodiments of the antibody-drug conjugate (ADC) of formula (Ia) or (Ib), CTX is selected from tubulin stabilizers, tubulin destabilizers, DNA alkylating agents, DNA minor groove binding agents, DNA intercalating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, gyrase inhibitors, protein synthesis inhibitors, proteosome inhibitors, and antimetabolites.

[0270] In some embodiments of the antibody-drug conjugate (ADC) of formula (Ia) or (Ib), CTX is a chemotherapeutic agent. Suitable chemotherapeutic agents will be known to those skilled in the art, such as those disclosed in Chu. E., Devite, VT, 2012, Physician's Cancer Chemistry Drug Manuscript, 2012 (Jones & Bartlett Learning Oncology), and similar literature.

[0271] In some implementations, CTX can be any FDA-approved chemotherapy agent. In some implementations... In this protocol, CTX can be any FDA-approved chemotherapy agent used for cancer treatment.

[0272] In some embodiments, the CTX is selected from alkylating agents, anthracyclines, cytoskeleton disruptors (taxanes), epothilone, histone deacetylase inhibitors (HDAC), inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, monoclonal antibodies, nucleotide analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids or derivatives thereof, and radioisotopes.

[0273] In some embodiments, the CTX is selected from actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, docetaxel, deoxyfluorouridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, methyldichlorodiethylamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, penoxorubicin, vincristine, vinblastine, vindesine, and vinorelbine.

[0274] In some embodiments, the CTX is selected from tubulin stabilizers, tubulin destabilizers, DNA alkylating agents, DNA minor groove binding agents, DNA intercalating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, gyrase inhibitors, protein synthesis inhibitors, proteosome inhibitors, and antimetabolites.

[0275] In some embodiments, the CTX is selected from actinomycin D, aminonaphthylfentanyl, auristatin, benzophenone, benzothiazole, chalcogenine, camptothecin, CC-1065 (NSC298223), cimadolicine, colchicine, cobustatin A4, dolastatin, doxorubicin, elenefad, emtansine (DM1), etoposide KF-12347 (leinamycin), maytansine derivatives, methotrexate, mitoxantrone, nocodazole, protein body inhibitor 1 (PSI1), baculosporin A, T-2 toxin (a trichoderma analog), paclitaxel, tubulysin, Velcade®, and vincristine. In some embodiments, the CTX is auristatin, chalcogenine, maytansine derivatives, or tubulysin.

[0276] In some embodiments, the CTX is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), pyrrolobenzodiazepine (PDB), chalcogenin γ, mertansine, or tubulin T2. ​​In some embodiments, the CTX is MMAE or MMAF. In some embodiments, the CTX is PDB. In some embodiments, the CTX is tubulin T2. ​​In some embodiments, the CTX is tubulin T3 or tubulin T4, the structure of which is provided below:

[0277] 5.3 Other molecules that bind to BTN1A1 or its ligands On the other hand, this document provides molecules that selectively bind to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA), thereby inhibiting the binding of BTN1A1 ligands to BTN1A1. In some embodiments, the molecule is not an antibody and does not include an antigen-binding domain. In some embodiments, the molecule is a decoy receptor, such as a GAL-1, GAL-9, NRP-2, BTLA, or BTN1A1 decoy receptor or a soluble receptor. In some embodiments, the molecule can inhibit the formation of BTN1A1-BTN1A1 ligand complexes (e.g., complexes comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1A1). In some embodiments, the molecule can disrupt the formation of BTN1A1-BTN1A1 ligand complexes (e.g., complexes comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1A1). In some embodiments, the molecules provided herein that selectively bind BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) can inhibit the binding of BTN1A1 ligands to BTN1A1, thereby inhibiting the immunosuppressive function of BTN1A1 or BTN1A1-BTN1A1 ligand complexes.

[0278] On the other hand, this document provides molecules that selectively bind to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA), thereby inhibiting the binding of BTN1A1 ligands to BTN1A1. In some embodiments, the molecule is not an antibody and does not have an antigen-binding fragment. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-1. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-9. In some embodiments, the molecule inhibits the binding of BTN1A1 to NRP-2. In some embodiments, the molecule inhibits the binding of BTN1A1 to BTLA. In some embodiments, the molecule completely inhibits the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can at least partially inhibit the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the binding of BTN1A1 to BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or its inhibition is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of BTN1A1 to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) with an IC50 value less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some implementations, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

[0279] In some embodiments, the molecule selectively binds to BTN1A1, thereby inhibiting the binding of two or more BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and GAL-9 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of NRP-2 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A1.

[0280] In some implementations, the molecule selectively binds to the extracellular domain (ECD) of BTN1A1.

[0281] In some embodiments, the molecules provided herein can bind to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) with dissociation constants of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 2080 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the molecule can bind to GAL-1 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the molecule can bind to Gal-9 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the molecule may bind to NRP-2 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.In some embodiments, the molecule may bind to BTLA with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.

[0282] In some embodiments, the molecule can modulate the activity or signal transduction of BTN1A1, or modulate the activity or signal transduction of a complex of BTN1A1 and a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA.

[0283] In some embodiments, the molecule can regulate T cell activity. In some embodiments, the T cells are CD8+ cells. In some embodiments, the molecule can increase T cell activation or T cell proliferation. In some embodiments, the molecule can inhibit T cell apoptosis.

[0284] In some embodiments, the molecule is a GAL-1 decoy. In some embodiments, the molecule is a GAL-1 decoy as described in International Patent Application PCT / US2002 / 031273 (e.g., disclosed as WO2003026494A3), which is incorporated herein by reference. In some embodiments, the molecule is a GAL-9 decoy. In some embodiments, the molecule is an NRP-2 decoy. In some embodiments, the molecule is a BTLA decoy receptor. In some embodiments, the molecule is a soluble BTLA receptor (e.g., a BTLA extracellular domain construct, such as a BTLA-ECD-Fc construct). In some embodiments, the molecule is a membrane-bound BTLA decoy receptor (e.g., a truncated BTLA lacking a cytoplasmic domain). In some embodiments, the molecule is a BTN1A1 decoy or a soluble receptor. In some embodiments, the molecule is a BTN1A1 binder, as described in International Application No. PCT / US20104 / 071853 (e.g., disclosed as WO2010100219A1).

[0285] 5.4 Composition This document also provides compositions having molecules that selectively bind to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby said molecules inhibit the binding of BTN1A1 ligands to BTN1A1. In some embodiments, said molecules are not antibodies and do not include antigen-binding fragments. In some embodiments, said molecules are decoy receptors, such as GAL-1, GAL-9, NRP-2, BTLA, or BTN1A1 decoy receptors or soluble receptors.

[0286] On the other hand, this document also provides compositions comprising an antigen-binding fragment molecule that immunely and specifically binds to BTN1A1 or a BTN1A1 ligand, thereby inhibiting the binding of a BTN1A1 ligand (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A. In some embodiments, the antigen-binding fragment immunely and specifically binds to BTN1A (e.g., glycosylated BTN1A or a BTN1A dimer). In some embodiments, the antigen-binding fragment immunely and specifically binds to a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). In some embodiments, the antigen-binding fragment immunely and specifically binds to GAL-1. In some embodiments, the antigen-binding fragment immunely and specifically binds to GAL-9. In some embodiments, the antigen-binding fragment immunely and specifically binds to NRP-2. In some embodiments, the antigen-binding fragment immunely and specifically binds to BTLA. In some embodiments, the antigen-binding fragment immunely and specifically binds to BTN1A. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55, N215, and / or N449. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at position N55. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at position N215. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at position N449. In some embodiments, the antigen-binding fragment binds specifically to one or more glycosylated motifs. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55 and N215. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N215 and N449. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55 and N449. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 glycosylated at positions N55, N215, and N449. In some embodiments, the antigen-binding fragment binds specifically to BTN1A1 dimers, such as one or more glycosylated BTN1A1 dimers at positions N55, N215, and N449 of one or more BTN1A1 monomers in a BTN1A dimer.

[0287] In some embodiments, the composition comprises a molecule having an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-1. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-9. In some embodiments, the molecule inhibits the binding of BTN1A1 to NRP-2. In some embodiments, the molecule inhibits the binding of BTN1A1 to BTLA. In some embodiments, the molecule completely inhibits the binding of BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule at least partially inhibits the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1A1 to a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). In some embodiments, the binding of BTN1A1 to a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or its inhibition is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of BTN1A1 to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) with an IC50 value less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance assay, a biolayer interference assay, a co-immunoprecipitation assay, a FRET or TR-FRET assay, or an ELISA.

[0288] In some embodiments, the composition comprises a molecule having an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of two or more BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and GAL-9 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of NRP-2 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN1A. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN1A. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A.

[0289] In some embodiments, the composition comprises a molecule having an antigen-binding fragment that specifically binds to a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), thereby inhibiting the binding of the BTN1A ligand to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to GAL-1, and the molecule inhibits the binding of GAL-1 to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to GAL-9, and the molecule inhibits the binding of GAL-9 to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to NRP-2, and the molecule inhibits the binding of NRP-2 to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to BTLA, and the molecule inhibits the binding of BTLA to BTN1A1. In some embodiments, the molecule can completely inhibit the binding of a BTN1A1 ligand (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can at least partially inhibit the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the binding of BTN1A1 to BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or its inhibition is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of BTN1A1 to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) with IC50 values ​​less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM.In some implementations, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

[0290] In some embodiments, this document provides compositions having molecules having an antigen-binding fragment capable of binding to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) with dissociation constants of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less, whereby the molecule is capable of inhibiting the binding of BTN1A1 ligands to BTN1A1. In some embodiments, the antigen-binding fragment may bind to GAL-1 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the antigen-binding fragment may bind to GAL-9 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the antigen-binding fragment may bind to NRP-2 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.In some embodiments, the antigen-binding fragment may bind to BTLA with a binding dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.

[0291] In some embodiments, the molecules in the compositions provided herein have an antigen-binding fragment that specifically binds to BTN1A1, wherein the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the glycosylated BTN1A1 is a BTN1A1 dimer. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55, N215, and / or N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at position N55 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at position N215 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at position N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to one or more glycosylation motifs. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55 and N215 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N215 and N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55 and N449 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 glycosylated at positions N55, N215, and N449 relative to non-glycosylated BTN1A1.

[0292] In some embodiments, the molecules in the compositions provided herein have an antigen-binding fragment that specifically binds to BTN1A1, wherein the antigen-binding fragment preferentially binds to the BTN1A1 dimer relative to the BTN1A1 monomer. In some embodiments, one or more of the BTN1A1 monomers in the BTN1A1 dimer are glycosylated at one or more of positions N55, N215, and N449.

[0293] In some embodiments, the molecules in the compositions provided herein have an antigen-binding fragment that binds to K+ of glycosylated BTN1A1. D Compared to the K-type expression exhibited when bound to non-glycosylated BTN1A1. D Half as much. In some embodiments, the antigen-binding fragment binds to K-type glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least twice as small. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 5 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 10 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 15 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 20 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 25 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 30 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. D Compared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 40 times smaller. In some embodiments, the antigen-binding fragment binds to K+ of glycosylated BTN1A1. DCompared to the K exhibited by binding with non-glycosylated BTN1A1 D At least 50 times smaller.

[0294] In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D Compared to binding with BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers), exhibiting K D Half the size. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least twice as small. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 5 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 10 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 15 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 20 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 25 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) DAt least 30 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 40 times smaller. In some embodiments, the antigen-binding fragment binds to the K-molecule of the BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer). D The K exhibited when bound to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers) D At least 50 times smaller.

[0295] In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least twice that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least five times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least ten times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least 15 times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least 20 times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least 25 times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least 30 times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least 40 times that binding to non-glycosylated BTN1A1. In some embodiments, the MFI of the antigen-binding fragment binding to glycosylated BTN1A1 is at least 50 times that binding to non-glycosylated BTN1A1.

[0296] In some embodiments, the MFI of the antigen-binding fragment binding to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least twice as high as the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least five times as high as the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least ten times as high as the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least 15 times higher than the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least 20 times higher than the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least 25 times higher than the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) is at least 30 times higher than the MFI exhibited with a BTN1A1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) is at least 35 times higher than the MFI exhibited with a BTN1A1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment binding to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) is at least 40 times higher than the MFI exhibited with a BTN1A1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the MFI of the antigen-binding fragment bound to the BTN1A dimer (e.g., glycosylated BTN1A dimer) is at least 50 times greater than the MFI exhibited with the BTN1A1 monomer (e.g., glycosylated BTN1A monomer).

[0297] In another aspect, this document provides a composition having a molecule having a BTN1A1 glycosylated antigen-binding fragment at immune-specific masking sites N55, N215, and / or N449. In some embodiments, the BTN1A1 at the immune-specific masking site N55 of the antigen-binding fragment is glycosylated. In some embodiments, the BTN1A1 at the immune-specific masking site N215 of the antigen-binding fragment is glycosylated. In some embodiments, the BTN1A1 at the immune-specific masking site N449 of the antigen-binding fragment is glycosylated. In some embodiments, the antigen-binding fragment immune-specifically masks one or more glycosylation motifs of BTN1A1. In some embodiments, the BTN1A1 at immune-specific masking sites N55 and N215 of the antigen-binding fragment is glycosylated. In some embodiments, the BTN1A1 at immune-specific masking sites N215 and N449 of the antigen-binding fragment is glycosylated. In some embodiments, BTN1A1 is glycosylated at immune-specific masking sites N55 and N449 of the antigen-binding fragment. In some embodiments, BTN1A1 is glycosylated at immune-specific masking sites N55, N215, and N449 of the antigen-binding fragment.

[0298] In some embodiments, the composition may have at least 0.1% by weight of the antibody or other molecules described herein. In some embodiments, the composition may have at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more by weight of an anti-BTN1A1 antibody, an anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody, or other molecules having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand. In other embodiments, for example, the anti-BTN1A1 antibody, anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or other molecules having an immune-specific binding fragment to BTN1A1 may comprise about 2% to about 75%, about 25% to about 60%, about 30% to about 50%, or any range thereof, of the composition by weight.

[0299] The composition may be a pharmaceutical composition having an anti-BTN1A1 antibody, an anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-, or BTLA) antibody, or other antigen-binding fragments that specifically bind to BTN1A1 or BTN1A1 ligands as active ingredients, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also include one or more additional active ingredients. A pharmaceutically acceptable carrier may be a carrier approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopoeia for use in animals, and more specifically in humans.

[0300] As per this disclosure, the preparation of pharmaceutical compositions having antibodies or other molecules as active ingredients as described herein is known to those skilled in the art, as illustrated in Remington's Pharmaceutical Sciences, 18th edition, 1990, which is incorporated herein by reference. Furthermore, for administration to animals (including humans), it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Biostandards Agency.

[0301] Pharmaceutically acceptable carriers include liquids, semi-solids (i.e., pastes), or solid carriers. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, or combinations thereof. The pharmaceutically acceptable carrier may include aqueous solvents (e.g., water, alcohol / aqueous solutions, ethanol, saline solutions, parenteral carriers such as sodium chloride, Ringer's glucose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delay agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids such as glycine and lysine, carbohydrates such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol), Materials such as gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquids, and nutrient supplements, and combinations thereof, are known to those skilled in the art. The use of any conventional medium, reagent, diluent, or carrier in an applicable composition for practicing this method is appropriate unless it is detrimental to the recipient or to the therapeutic efficacy of the composition therein. The pH value and the exact concentrations of the various components in the pharmaceutical composition are adjusted according to known parameters. According to certain aspects of the present disclosure, the composition can be combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, grinding, etc. Such processes are routine to those skilled in the art.

[0302] In some embodiments, a pharmaceutically acceptable carrier may be a pH-buffered aqueous solution. Examples include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (e.g., fewer than about 10 amino acid residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN. TM Polyethylene glycol (PEG) and PLURONICS TM .

[0303] In some embodiments, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water can be a carrier, especially when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, calcium carbonate, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, polysorbate-80, etc. The compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0304] Pharmaceutically acceptable carriers include liquids, semi-solids (i.e., pastes), or solid carriers. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, or combinations thereof. The pharmaceutically acceptable carrier may include aqueous solvents (e.g., water, alcohol / aqueous solutions, ethanol, saline solutions, parenteral carriers such as sodium chloride, Ringer's glucose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delay agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids such as glycine and lysine, carbohydrates such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol), Materials such as gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, liquids, and nutrient supplements, and combinations thereof, are known to those skilled in the art. The use of any conventional medium, reagent, diluent, or carrier in an applicable composition for practicing this method is appropriate unless it is detrimental to the recipient or to the therapeutic efficacy of the composition therein. The pH value and the exact concentrations of the various components in the pharmaceutical composition are adjusted according to known parameters. According to certain aspects of the present disclosure, the composition can be combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, grinding, etc. Such processes are routine to those skilled in the art.

[0305] The anti-BTN1A1 antibody, anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, RP-, or BTLA) antibody, or other molecules having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand, can be formulated into compositions in the form of a free base, neutral, or salt. Pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with the free amino group of a protein composition, or acid addition salts formed with an inorganic acid, such as hydrochloric acid or phosphoric acid, or acid addition salts formed with an organic acid, such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts forming a free carboxyl group can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide; or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, or procaine.

[0306] In further embodiments, pharmaceutical compositions comprising lipids are provided herein. Lipids can broadly include a class of substances characterized by being insoluble in water and extractable with organic solvents. Examples include compounds containing long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., designed or produced by humans). Lipids can be biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, glycerol phosphates, steroids, terpenes, lysophospholipids, glycosphingolipids, glycolipids, sulfates, lipids of fatty acids having ether and ester linkages, polymerizable lipids, and combinations thereof. Compounds other than those specifically described herein that are understood by those skilled in the art to be lipids may also be used.

[0307] Those skilled in the art will be familiar with a range of techniques that can be used to disperse compositions in lipid carriers. For example, antibodies can be dispersed in a lipid-containing solution, dissolved in lipids, emulsified with lipids, mixed with lipids, bound to lipids, covalently bound to lipids, contained in lipids as a lipid suspension, comprising micelles or liposomes, or complexed with micelles or liposomes, or bound to lipids or lipid structures by any method known to those skilled in the art. Such dispersion may or may not result in the formation of liposomes.

[0308] Typically, the components of the composition are supplied either individually or mixed together in unit dosage forms, for example, in sealed containers such as ampoules or sachets indicating the quantity of active reagent, as lyophilized powders or anhydrous concentrates. When the composition is administered via infusion, it can be reconstituted with an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be provided to mix the components prior to administration.

[0309] The amount of active ingredient in each therapeutically useful composition can be prepared in such a way that a suitable dose is obtained from any given unit dose of the compound. Those skilled in the art will recognize the need for various dosages and treatment regimens due to factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations.

[0310] A unit dose refers to a physically separated unit applicable to the subject, each unit containing a predetermined quantity of the pharmaceutical composition, calculated to produce the desired response discussed above when associated with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered depends on the desired effect, depending on the number of treatments and the unit dose. The number of reagent doses of the composition of this embodiment administered to the patient or subject can be determined by physical and physiological factors such as weight, age, health, subject's sex, type of disease to be treated, degree of disease penetration, previous or concurrent treatment interventions, patient's spontaneous illness, route of administration, and the potency, stability, and toxicity of the specific therapeutic substance. In other non-limiting instances, the dosage may be approximately 1 microgram / kg / body weight, approximately 5 micrograms / kg / body weight, approximately 10 micrograms / kg / body weight, approximately 50 micrograms / kg / body weight, approximately 100 micrograms / kg / body weight, approximately 200 micrograms / kg / body weight, approximately 350 micrograms / kg / body weight, approximately 500 micrograms / kg / body weight, approximately 1 mg / kg / body weight, approximately 5 mg / kg / body weight, approximately 10 mg / kg / body weight, approximately 50 mg / kg / body weight, approximately 100 mg / kg / body weight, approximately 200 mg / kg / body weight, approximately 350 mg / kg / body weight, approximately 500 mg / kg / body weight, up to approximately 1000 mg / kg / body weight or higher, and any range that may be derived therefrom. In non-limiting examples of the ranges that can be derived from the figures listed herein, ranges such as approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight, etc., can be used, based on the figures described above. In any case, the physician administering the medication will determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

[0311] Those skilled in the art will understand that the compositions described herein are not limited to the specific properties of therapeutic articles. For example, such compositions may be provided in formulations with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic articles may be administered to mammals for veterinary use, e.g., for livestock, and for clinical use in humans in a manner similar to other therapeutic agents. Generally, the dosage required for therapeutic efficacy varies depending on the type of application and route of administration, as well as the specific needs of the individual subject. The actual dose of a composition administered to animal patients, including human patients, may be determined by physical and physiological factors, such as body weight, severity of condition, type of disease to be treated, prior or concurrent therapeutic interventions, the patient's spontaneous disease, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of times an effective amount is administered may vary depending on the subject's response. In any case, the physician administering the medication will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.

[0312] 5.5 Therapeutic Uses and Treatment Methods BTN1A1 is specifically and highly expressed in cancer cells.

[0313] On the other hand, this document provides the therapeutic use of molecules that selectively bind to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) in cancer treatment, wherein said molecules are capable of inhibiting the binding of BTN1A1 ligands to BTN1A1. In some embodiments, said molecules are not antibodies and do not include antigen-binding fragments. In some embodiments, said molecules are decoy receptors, such as GAL-1, GAL-9, NRP-2, BTLA, or BTN1A1 decoy receptors or soluble receptors.

[0314] On the other hand, this document provides the therapeutic use of molecules having immune-specific binding fragments to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antigen-binding fragments in cancer treatment, whereby said molecules inhibit the binding of BTN1A1 ligands to BTN1A1. In some embodiments, these molecules bind to cancer cells expressing BTN1A1 and induce an immune response leading to the destruction of these cancer cells. In some embodiments, said molecules inhibit BTN1A1-BTN1A1 ligand (e.g., GAL-1, GAL-2, NRP-2, BTLA) complexes. For example, in some embodiments, said molecules prevent the formation of BTN1A1-BTN1A1 ligand complexes or disrupt existing BTN1A1-BTN1A1 ligand complexes. The molecules provided herein can enhance T-cell-dependent apoptosis in cancer cells and inhibit cancer cell proliferation.

[0315] On the other hand, the present invention provides a method for treating a cancer subject, comprising administering to the subject a therapeutically effective amount of a molecule that selectively binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby said molecule inhibits the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, said molecule is not an antibody and does not include an antigen-binding fragment. In some embodiments, said molecule is a decoy receptor, such as a GAL-1, GAL-9, NRP-2, BTLA, or BTN1A1 decoy receptor or a soluble receptor.

[0316] On the other hand, the present invention provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (such as GAL-1, GAL-9, NRP-2, or BTLA), thereby said molecule inhibiting the binding of the BTN1A1 ligand BTN1A1.

[0317] In some embodiments, the molecule has an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-1. In some embodiments, the molecule inhibits the binding of BTN1A1 to GAL-9. In some embodiments, the molecule inhibits the binding of BTN1A1 to NRP-2. In some embodiments, the molecule inhibits the binding of BTN1A1 to BTLA. In some embodiments, the molecule can completely inhibit the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can at least partially inhibit the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the binding of BTN1A1 to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) or its inhibition is determined using surface plasmon resonance biolayer interferometry or co-immunoprecipitation. In some embodiments, the molecule can inhibit the binding of BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1 with IC50 values ​​less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some implementations, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

[0318] In some embodiments, the molecule has an antigen-binding fragment that specifically binds to BTN1A1, thereby inhibiting the binding of two or more BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and GAL-9 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of NRP-2 and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and NRP-2 to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN1A1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A1.

[0319] In some embodiments, the molecule has an antigen-binding fragment that specifically binds to a BTN1A1 ligand (such as GAL-1, GAL-9, NRP-2, or BTLA), thereby enabling the molecule to inhibit the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to GAL-1, and the molecule inhibits GAL-1 binding to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to GAL-9, and the molecule inhibits GAL-9 binding to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to NRP-2, and the molecule inhibits NRP-2 binding to BTN1A1. In some embodiments, the antigen-binding fragment specifically binds to BTLA, and the molecule inhibits BTLA binding to BTN1A1. In some embodiments, the molecule can completely inhibit the binding of the BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can at least partially inhibit the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the molecule can inhibit the binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1. In some embodiments, the binding of BTN1A1 to BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or its inhibition is determined using surface plasmon resonance, biolayer interference, or co-immunoprecipitation. In some embodiments, the molecule can inhibit the binding of BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A1 with IC50 values ​​less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance assay, a biolayer interference assay, a co-immunoprecipitation assay, a FRET or TR-FRET assay, or an ELISA.

[0320] In some embodiments, the molecule has an antigen-binding fragment capable of binding to BTN1A1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less, thereby enabling the molecule to inhibit the binding of BTN1A1 ligands to BTN1A1. In some embodiments, the antigen-binding fragment may bind to GAL-1 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the antigen-binding fragment may bind to GAL-9 with a dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less. In some embodiments, the antigen-binding fragment may bind to NRP-2 with a binding dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.In some embodiments, the antigen-binding fragment may bind to BTLA with a binding dissociation constant of 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 3 nM or less, or 1 nM or less.

[0321] In some embodiments, the molecules provided herein have an antigen-binding fragment that specifically binds to BTN1A1, including an anti-BTN1A1 antibody that causes BTN1A1 internalization into lysosomes. Therefore, this document also provides a method for delivering a compound to cells expressing BTN1A1 by contacting cells with a compound-conjugated molecule provided herein using the molecules provided herein. The compound may be an imaging agent, therapeutic agent, toxin, or radionuclide as described herein. The compound may be conjugated to an anti-BTN1A1 antibody. The conjugate may be any conjugate described herein, such as an ADC. The cells may be cancer cells. The cells may also be a cell population comprising both cancer cells and normal cells. Because cancer cells specifically and highly express BTN1A1, the molecules described herein can be used to achieve specific drug delivery to cancer cells rather than normal cells.

[0322] In some embodiments, the molecules provided herein, including anti-BTN1A1 antibodies and anti-BTN1A1 ligand (GAL-1, GAL-9, NRP-2, BTLA) antibodies, can modulate the immune response of a subject. In some embodiments, the molecules can promote T cell activation. In some embodiments, the molecules can promote T cell proliferation. In some embodiments, the molecules can increase cytokine production. In some embodiments, the molecules provided herein can also enhance T cell-dependent apoptosis of BTN1A1-expressing cells or inhibit the proliferation of BTN1A1-expressing cells.

[0323] Therefore, this article provides a method for modulating the immune response of a subject by administering an effective amount of a molecule provided herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), capable of inhibiting the binding of BTN1A1 ligands to BTN1A1, including anti-BTN1A1 antibodies or anti-BTN1A1 ligand antibodies. Modulation of the immune response may include (a) increasing T cell activation (e.g., CD8+). +(b) increase T cell activation; and / or (c) increase cytokine production. In some embodiments, the method further includes administering anti-PD1 therapy or anti-PD-L1 therapy.

[0324] This document also provides a method for enhancing T-cell-dependent apoptosis of BTN1A1-expressing cells by contacting an effective amount of the molecule described herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), including an anti-BTN1A1 antibody or an anti-BTN1A1 ligand antibody, thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. This document also provides a method for inhibiting cell proliferation by contacting BTN1A1-expressing cells with an effective amount of the molecule described herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), including an anti-BTN1A1 antibody, thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. The cells may be cancer cells. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0325] In some embodiments, these molecules can be used to treat cancer by inhibiting the inhibitory activity of BTN1A1 in T cell activation or proliferation. Therefore, this document provides the use of these molecules in the immune system of a subject by inhibiting or blocking BTN1A1 signaling. In some embodiments, this document provides the use of these molecules to block BTN1A1 binding to T cells.

[0326] In some embodiments, these molecules cause destruction of cancer cells via ADCC or CDC mechanisms. In some embodiments, these molecules are engineered to have enhanced ADCC activity. In some embodiments, these molecules are engineered to have enhanced CDC activity. For example, these molecules can be engineered to have enhanced interactions with killer cells carrying Fc receptors. Methods for producing such engineered molecules are described herein and are known in the art.

[0327] On the other hand, the present invention provides a method for killing or inhibiting the proliferation of cancer cells resistant to PD-1 therapy or anti-PD-L1 therapy, comprising contacting the cells with an effective amount of a molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), thereby inhibiting the binding of BTN1A1 or the BTN1A1 ligand.

[0328] In some embodiments, this document provides the use of molecules having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in treating a disease or condition in a subject that overexpresses BTN1A1, wherein said molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, including anti-BTN1A1 antibodies and anti-BTN1A1 ligand antibodies. In some embodiments, the expression level of BTN1A1 in the subject is higher than a reference level. The reference level may be the average or intermediate expression level of BTN1A1 in a population of healthy individuals. The reference level may also be determined by statistical analysis of the expression levels in a sample population.

[0329] On the other hand, the present invention provides a method for treating a subject with anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer, comprising administering to the subject a therapeutically effective amount of a molecule, said molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1.

[0330] In some embodiments, the subject has cancer resistant to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has cancer resistant to anti-PD-1 therapy. In some embodiments, the subject has cancer resistant to anti-PD-L1 therapy.

[0331] In some embodiments, the subject has cancer that is refractory to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has cancer that is refractory to anti-PD-1 therapy. In some embodiments, the subject has cancer that is refractory to anti-PD-L1 therapy.

[0332] In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy comprises an anti-PD-1 or anti-PD-L1 antibody or antibody fragment, or a soluble PD-1 or PD-L1 ligand, or its Fc-fusion protein (e.g., AMP-224, PD-L2Fc fusion soluble receptor).

[0333] In some implementations, the anti-PD-1 therapy includes nivolumab (opdivo), pembrolizumab (Keytruda®), pidizumab, AMP-514, or AMP-224.

[0334] In some implementations, the anti-PD-1 treatment includes the anti-PD-1 antibody provided in international application PCT / US20126 / 64394.

[0335] In some implementations, the anti-PD-L1 treatment includes YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0336] In some embodiments, the anti-PD-L1 treatment includes antibodies provided in international application No. PCT / US2016 / 024691, disclosed as WO2016 / 160792A1 and international application No. PCT / US2017 / 024027.

[0337] In some embodiments, the subject is treatment-naïve (e.g., the subject has not received any anticancer therapy) prior to treatment with a molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the subject has received one or more anticancer therapies other than antiPD-1 therapy or antiPD-L1 therapy (e.g., chemotherapy, radiation therapy, surgery, or with another targeted anticancer drug, such as Herceptin) prior to treatment with a molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). ® (Trastuzumab treatment). In some embodiments, the subject received one or more anti-PD1 or anti-PD-L1 therapies prior to molecular treatment with an antigen-binding fragment comprising an immune-specific binding fragment to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA).

[0338] In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer is lung cancer or breast cancer. In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer is lung cancer. In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer is breast cancer. In some embodiments, the lung cancer is Lewis lung cancer. In some embodiments, the breast cancer is breast cancer.

[0339] In some embodiments, the molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) is administered parenterally. In some embodiments, the molecule comprises an anti-BTN1A1 dimer antibody or an antigen-binding fragment thereof.

[0340] In some implementations, the treatment produces at least one therapeutic effect, such as reducing tumor size, reducing the number of metastatic lesions over time, achieving a complete response, a partial response, or stabilizing the disease.

[0341] On the other hand, this article provides a method for treating cancer, comprising (i) obtaining a sample including cancer cells from a subject suffering from cancer; (ii) determining the level of BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in the sample; and (iii) diagnosing the cancer if the level of BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in the sample is higher than or equal to a reference level of BTN1A1 or a BTN1A1 ligand. The subject may be responsive to molecular therapy comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), and (iv) administer to the subject a therapeutically effective amount of the molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit the binding of the BTN1A1 ligand to BTN1A1.

[0342] On the other hand, this article provides a method for treating cancer, comprising (i) obtaining a sample including cancer cells from a subject suffering from cancer; (ii) determining the level of PD-L1 in the sample; (iii) diagnosing the subject as potentially responsive to molecular therapy comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits the binding of the BTN1A1 ligand to BTN1A1; and (iv) administering to the subject a therapeutically effective amount of the molecule comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand.

[0343] On the other hand, this article provides a method for treating cancers resistant or refractory to anti-PD1 therapy or anti-PD-L1 therapy, comprising (i) obtaining a sample including cancer cells from a subject suffering from cancers resistant or refractory to anti-PD1 therapy or anti-PD-L1 therapy; (ii) determining the level of BTN1A1 or BTN1A1 ligands (GAL-1, GAL-9, NRP-2, BTLA) in the sample; (iii) diagnosing the subject as potentially responsive to molecular therapy comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits the binding of the BTN1A1 ligand to BTN1A1 if the level of the BTN1A1 or BTN1A1 ligand in the sample is higher than or equal to a reference level of BTN1A1; and (iv) administering a therapeutically effective amount of the molecule to the subject, the molecule comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand.

[0344] On the other hand, this article provides a method for treating anti-PDL therapy or anti-PD-L1 therapy-resistant or refractory cancers, comprising (i) obtaining a sample including cancer cells from a subject suffering from anti-PD1 therapy or anti-PD-L1 therapy-resistant or refractory cancers; (ii) determining the level of BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA) and / or PD-L1 in the sample; and (iii) if the level of BTN1A1 or BTN1A1 ligands in the sample is higher than or equal to a reference level of BTN1A1 or BTN1A1. The diagnosis is made if the level of PD-L1 in the sample is equal to or lower than a reference level of PD-L1, indicating that the subject may be responsive to molecular therapy comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits the binding of the BTN1A1 ligand to BTN1A1, and (iv) administering a therapeutically effective amount of the molecule to the subject, the molecule comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand.

[0345] On the other hand, this article provides a method for treating cancer, comprising (i) obtaining a sample including cancer cells from a subject suffering from cancer; (ii) determining the levels of BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA) and / or PD-L1 in the sample; (iii) diagnosing that the subject may be responsive to molecular therapy comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand, whereby the molecule inhibits the binding of BTN1A1 to the BTN1A1 ligand, if the level of BTN1A1 or BTN1A1 ligand in the sample is higher than or equal to a reference level of BTN1A1 and / or if the level of PD-L1 in the sample is equal to or lower than a reference level of PD-L1, and (iv) administering a therapeutically effective amount of the molecule to the subject, the molecule comprising an antigen-binding fragment of an immune-specific BTN1A1 or BTN1A1 ligand.

[0346] In some embodiments, the subject has cancer that is resistant or refractory to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has cancer resistant to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has cancer resistant to anti-PD-1 therapy. In some embodiments, the subject has cancer resistant to anti-PD-L1 therapy. In some embodiments, the subject has cancer refractory to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has cancer refractory to anti-PD-1 therapy. In some embodiments, the subject has cancer refractory to anti-PD-L1 therapy.

[0347] In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer is lung cancer or breast cancer. In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer is lung cancer. In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy-resistant or refractory cancer is breast cancer. In some embodiments, the lung cancer is Lewis lung cancer. In some embodiments, the breast cancer is breast cancer.

[0348] In some implementations, the subject has cancer that is at least partially responsive to anti-PD-1 therapy or anti-PD-L1 therapy.

[0349] In some embodiments, BTN1A1 is expressed in the cancer. In some embodiments, the cancers expressing BTN1A1 include, for example, breast cancer, neuroendocrine prostate cancer (NEPC), diffuse large B-cell lymphoma, melanoma, cancers from the National Cancer Institute Cancer Panel (NCI60), uveal melanoma, pancreatic cancer, ovarian cancer, uterine cancer, lung adenocarcinoma, connective tissue proliferative small round cell tumor, bladder cancer, colorectal cancer, squamous cell carcinoma of the lung, liver cancer, lung cancer, stomach cancer, cholangiocarcinoma, esophageal squamous cell carcinoma, head and neck cancer, sarcoma, prostate cancer, liver cancer, pancreatic cancer, pheochromocytoma or paraganglioma (PCPG), cervical cancer, glioma, or acute myeloid leukemia (AML).

[0350] In some embodiments, the method includes determining the level of BTN1A1 or BTN1A1 ligand in the sample. In some embodiments, the method includes determining the level of PD-L1 in the sample. In some embodiments, the method includes determining the levels of BTN1A1 or BTN1A1 ligand and PD-L1 in the sample.

[0351] In some embodiments, if the level of BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample is higher than or equal to a reference level of BTN1A1 or a BTN1A1 ligand, the subject is diagnosed as potentially responsive to molecular therapy comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, Gal-9, NRP-2, BTLA), whereby the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, if the level of BTN1A1 or a BTN1A1 ligand in a sample is higher than a reference level of BTN1A1 or a BTN1A1 ligand, the subject is diagnosed as potentially responsive to molecular therapy comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand.

[0352] In some embodiments, if the PD-L1 level in a sample is below or equal to a reference PD-L1 level, the diagnostic subject may be responsive to molecular therapy comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, if the PD-L1 level in a sample is below a reference PD-L1 level, the diagnostic subject may be responsive to molecular therapy comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand.

[0353] In some implementations, if the level of BTN1A1 or BTN1A ligand in a sample is equal to or higher than a reference level for BTN1A1, and the level of PD-L1 is lower than or equal to a reference level for PD-L1, the diagnostic subject may be responsive to molecular therapy that includes an antigen-binding fragment comprising an immune-specific binding fragment of BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits the binding of the BTN1A1 ligand to BTN1A1. In some implementations, if the level of BTN1A1 or a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample is higher than or equal to a reference level for BTN1A1, and the level of PD-L1 is lower than a reference level for PD-L1, the diagnostic subject may be responsive to molecular therapy comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), said molecule which inhibits the binding of the BTN1A1 ligand to BTN1A1. In some implementations, if the level of BTN1A1 or BTN1A ligand in a sample is higher than a reference level for BTN1A1 and the level of PD-L1 is lower than a reference level for PD-L1, the diagnostic subject may be responsive to molecular therapy that includes an antigen-binding fragment that specifically binds to BTN1A1 or BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA), said molecule which inhibits the binding of BTN1A1 ligands to BTN1A1.

[0354] The sample can be any solid or liquid sample from the object.

[0355] In some embodiments, the sample is a liquid biopsy sample. In some embodiments, the sample used in the methods provided herein includes bodily fluids from the subject. Non-limiting examples of bodily fluids include blood (e.g., whole blood), plasma, amniotic fluid, aqueous humor, bile, cerumen, Cooper's fluid, preejaculate fluid, chyle, chyme, female ejaculate, interstitial fluid, lymph, menstrual fluid, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubricant, vomit, water, feces, internal bodily fluids (including cerebrospinal fluid surrounding the brain and spinal cord), synovial fluid, intracellular fluid (fluid inside cells), and vitreous humor (fluid in the eye). In some embodiments, the sample is a blood sample. The blood sample can be obtained using conventional techniques, such as those described in Innis et al., eds., PCR Protocols (Academic Press, 1990). White blood cells can be isolated from blood samples using conventional techniques or commercially available kits, such as the RosetteSep kit (SteinCell Technologies, Vancouver, Canada). Subpopulations of white blood cells, such as monocytes, B cells, T cells, granulocytes, or lymphocytes, can be further separated using conventional techniques, such as magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

[0356] In some embodiments, the sample is a solid biopsy sample. In some embodiments, the sample used in the current method includes biopsy material (e.g., tumor biopsy material). The biopsy material can be derived from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph nodes, hair, spleen, brain, breast, or other organs. Any biopsy technique known to those skilled in the art can be used to separate the sample from the object, such as open biopsy, closed biopsy, core biopsy, excisional biopsy, resection biopsy, or fine-needle aspiration biopsy.

[0357] In some embodiments, the sample is a paraffin-embedded, formaldehyde-fixed tissue sample. In some embodiments, the sample is a tissue section. In some embodiments, the sample is presented on a tissue array.

[0358] In some implementations, the level of a biomarker is measured by determining the protein level of the biomarker.

[0359] The levels of BTN1A1, BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA), or PD-L1 in a sample can be analyzed using any method known in the art. See, for example, section 5.7 (Companion Diagnostics). In some embodiments, determining the levels of BTN1A1, BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA), or PD-L1 in a sample may include analyzing the nucleic acid levels or protein levels of BTN1A1, BTN1A1 ligands, or PD-L1. The nucleic acid levels of BTN1A1, BTN1A1 ligands, or PD-L1 can be analyzed, for example, using polymerase chain reaction (PCR) methods (e.g., RT-PCR or Q-PCR), nucleic acid array-based methods (e.g., gene chips), or nucleic acid sequencing methods such as next-generation sequencing methods. BTN1A1, BTN1A1 ligand, or PD-L1 protein levels can be determined using methods such as Western blotting, ELISA, FACS, and immunohistochemistry. BTN1A1, BTN1A1 ligand, or PD-L1 levels can be determined in an absolutely quantitative manner (e.g., the weight of BTN1A1, BTN1A1 ligand, or PD-L1 per part by weight of tissue, or the molar amount per tissue volume or per volume of liquid sample). In some embodiments, BTN1A1, BTN1A1 ligand, or PD-L1 levels are determined in a relative or semi-quantitative manner (e.g., the relative intensity of BTN1A1 or PD-L1-specific staining in different regions of a tissue sample). BTN1A1, BTN1A1 ligand, or PD-L1 levels can be determined independently using the same or different methods. In some embodiments, relative BTN1A1, BTN1A1 ligand, or PD-L1 levels are determined in tissue sections of solid tumor samples using immunohistochemistry combined with fluorescence microscopy or bright-field microscopy.

[0360] In some implementations, determining the levels of BTN1A1, BTN1A1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA), and / or PD-L1 in a sample includes, for example, analyzing the expression of BTN1A1, BTN1A1 ligands, and / or PD-L1 on the cell surface using FACS assays or immunocytochemistry.

[0361] In some implementations, the treatment produces at least one therapeutic effect, such as a reduction in tumor size, a reduction in the number of metastatic lesions over time, a complete response, a partial response, or a stable disease.

[0362] In some embodiments, a reference is prepared using a control sample obtained from the subject, derived from the same source as the sample, prior to administration of a molecule comprising an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, a reference is prepared using a control sample obtained from a healthy subject without cancer, derived from the same source as the sample. In some embodiments, a reference is prepared using a control sample obtained from a group of healthy subjects without cancer, derived from the same source as the sample. In some embodiments, a reference is prepared using a control sample obtained from a second subject with cancer, derived from the same source as the sample. In some embodiments, a reference is prepared using a control sample obtained from a group of subjects with cancer, derived from the same source as the sample.

[0363] 5.4.1 Diseases and Conditions In some embodiments, this document provides the use of antibodies or other molecules to mediate increased production of cytokines such as IFN-γ. Therefore, this document provides the use of such antibodies or other molecules in the treatment of diseases and conditions that can be treated with cytokines, such as ovarian cancer and other forms of cancer. In some embodiments, this document provides the use of antibodies and other molecules in mediating increased production of T cells (e.g., CD8+). + Use in T cell activity or proliferation. Therefore, in some embodiments, the use of such antibodies and other molecules in treating diseases and conditions such as cancer that can be treated by increasing T cell activity or proliferation is provided. In some embodiments, the use of the antibodies or other molecules described herein is provided to simultaneously mediate increased T cell activity and increased T cell proliferation.

[0364] Upregulation of the immune system is particularly desirable in cancer treatment. Furthermore, BTN1A1 is specifically and highly expressed in cancer cells. The molecules described in this article can also bind to cancer cells and cause their destruction through direct cytotoxicity or via ADCC or CDC mechanisms. Therefore, this article provides a method for treating cancer. Cancer refers to a tumor or mass caused by abnormal, uncontrolled cell growth. Cancer can be primary or metastatic.

[0365] In some embodiments, this document provides a method for treating cancer by administering a molecularly binding fragment having an immune-specific binding fragment to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), said molecule inhibiting the binding of the BTN1A1 ligand to BTN1A1. The treatment method can be used for cancers including any malignant cell type, such as those found in solid tumors or hematologic cancers. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the pancreas, colon, cecum, esophagus, stomach, brain, head, neck, thyroid, thymus, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic cancers include, but are not limited to, bone marrow tumors, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. In some embodiments, the method further includes administering anti-PD1 therapy or anti-PD-L1 therapy.

[0366] In some embodiments, this document provides a method of administering a molecularly therapeutic target cancer, the molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, wherein the cancer may be breast cancer, neuroendocrine prostate cancer (NEPC), diffuse large B-cell lymphoma, melanoma, cancer from the National Cancer Institute Cancer Panel (NCI60), uveal melanoma, pancreatic cancer, ovarian cancer, uterine cancer, lung adenocarcinoma, connective tissue proliferative small round cell tumor, bladder cancer, colorectal cancer, lung squamous cell carcinoma, liver cancer, lung cancer, stomach cancer, cholangiocarcinoma, esophageal squamous cell carcinoma, head and neck cancer, sarcoma, prostate cancer, liver cancer, pancreatic cancer, pheochromocytoma or paraganglioma (PCPG), cervical cancer, glioma, or acute myeloid leukemia (AML). Molecules used for cancer treatment can be any molecule described herein that has an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at positions N55, N215, N449, or any combination thereof.

[0367] In some embodiments, this document provides a method of treating target cancer by administering a molecule described herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein said molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, wherein said cancer may be squamous cell carcinoma of the lung, adenocarcinoma of the prostate, adenocarcinoma of the pancreas, or hepatocellular carcinoma. The molecule for treating squamous cell carcinoma of the lung, adenocarcinoma of the prostate, adenocarcinoma of the pancreas, or hepatocellular carcinoma may be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand, thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, said antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1A1 glycosylation at positions N55, N215, N449, or any combination thereof.

[0368] In some embodiments, this document provides a method of treating target cancer by administering a molecule described herein, the molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1, wherein the cancer is a PD-1 or PD-L1 resistant or refractory cancer. The molecule for treating PD-1 or PD-L1 resistant or refractory cancer can be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at positions N55, N215, N449, or any combination thereof. In some embodiments, the cancer is anti-PD-1 therapy resistant or refractory cancer. In some embodiments, the cancer is anti-PD-L1 therapy resistant or refractory cancer. In some embodiments, the anti-PD-1 therapy resistant or refractory cancer is breast cancer or lung cancer. In some embodiments, the anti-PD-1 therapy resistant or refractory cancer is breast cancer or Lewis lung cancer. In some embodiments, the anti-PD-1 therapy resistant or refractory cancer is breast cancer. In some embodiments, the anti-PD-1 therapy resistant or refractory cancer is Lewis lung cancer.

[0369] Further examples of cancers that can be treated using the methods described in this article include, but are not limited to, malignant tumors, lymphomas, germ cell tumors, sarcomas, leukemia, squamous cell carcinoma, lung cancers (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, mesothelioma), peritoneal cancers, hepatocellular carcinomas, gastric cancers (including gastrointestinal cancers and gastrointestinal stromal carcinomas), esophageal cancers, pancreatic cancers, glioblastomas, cervical cancers, ovarian cancers, liver cancers, bladder cancers, breast cancers, colon cancers, colorectal cancers, endometrial or uterine cancers, salivary gland cancers, kidney cancers, prostate cancers, vulvar cancers, thyroid cancers, various types of head and neck cancers, melanomas, superficially spreading melanomas, spotted malignant melanomas, acral lentigines melanomas, and other cancers. Group melanoma, uveal melanoma, germ cell tumors (yolk sac tumors, testicular cancer, malignant syncytial tumors), and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleavage cell NHL; macromastopathy NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, multiple myeloma, acute myeloid leukemia (AML), and chronic myeloblastic leukemia.

[0370] The cancer can also be any of the following histological types: malignant growths; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatal carcinoma; metastatic cell carcinoma; papillary metastatic cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma of familial colonic polyps; solid carcinoma; malignant carcinoid tumor; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic granulocytic carcinoma; eosinophilic adenocarcinoma; basophilic granulocytic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-capsulated sclerosing carcinoma; adrenal gland Cortical carcinoma; Endometrioid carcinoma; Skin appendage carcinoma; Apocrine gland carcinoma; Sebaceous gland carcinoma; Succinate adenocarcinoma; Mucoepidermoid carcinoma; Cystic adenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous gland carcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant osteoblastoma; Podocyte carcinoma; Malignant testicular stromal cell tumor; Malignant lipocytoma; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Hemangioendothelioma; Malignant melanoma; Amelanoma; Superficially spreading melanoma; Malignant giant nevus Melanoma; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant stromal tumor; Malignant Brenner's ovarian tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonic carcinoma; Malignant teratoma; Malignant ovarian thyroid tumor; Malignant syncytial tumor; Malignant mesonephroma; Hemangioendothelioma; Malignant hemangioendothelioma; Multiple hemorrhagic sarcomas of the skin; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Supercortical osteosarcoma; Chondrosarcoma; Malignant chondrocyte Mesodermal chondrosarcoma; Giant cell tumor of bone; Ewing sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pineal tumor; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Plasmoblastoma; Fibroblastoma; Astrocytoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primary neuroectodermal; Cerebellar sarcoma; Ganglioblastoma; Neuroblastoma; Retinoblastoma; Oligosacral neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's syndrome; Granulomatoid; Small lymphocytic malignant lymphoma;Large cell, diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomatous leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myelosarcoma; and hairy cell leukemia.

[0371] In some embodiments, this document provides a method for treating a target cancer by administering the molecules described herein, said molecules having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein said molecules are capable of inhibiting the binding of BTN1A ligands to BTN1A1, wherein said cancer is lung cancer, prostate cancer, pancreatic cancer, ovarian cancer, liver cancer, head and neck cancer, breast cancer, or stomach cancer. In some embodiments, this document provides a method for treating a target cancer by administering the molecules described herein, said molecules having an antigen-binding fragment that specifically binds to BTN1A or a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein said molecules are capable of inhibiting the binding of BTN1A1 ligands to BTN1A1, wherein said cancer may be lung cancer. The lung cancer may be non-small cell lung cancer (NSCLC). The lung cancer may be small cell lung cancer (SCLC). The NSCLC may be squamous NSCLC. Molecules used to treat lung cancer can be any molecule described herein that has an antigen-binding fragment that specifically binds to BTN1A1 or glycosylated BTN1A. In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at positions N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0372] In some embodiments, this document provides a method of treating a target cancer by administering a molecule described herein, the molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, wherein the cancer may be prostate cancer. The molecule for treating prostate cancer may be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at sites N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0373] In some embodiments, this document provides a method of treating a target cancer by administering a molecule described herein, the molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, wherein the cancer may be pancreatic cancer. The molecule for treating pancreatic cancer may be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), thereby inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to a BTN1A1 dimer (e.g., a glycosylated BTN1A1 dimer) relative to a BTN1A1 monomer (e.g., a glycosylated BTN1A1 monomer). In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0374] In some embodiments, this document provides a method of treating a target cancer by administering a molecule described herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein said molecule is capable of inhibiting the binding of the BTN1A1 ligand to BTN1A1, whereby said cancer may be ovarian cancer. The molecule for treating ovarian cancer can be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby said molecule is capable of inhibiting the binding of the BTN1A1 ligand to BTN1A1. In some embodiments, said antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at sites N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0375] In some embodiments, this document provides a method of treating a target cancer by administering a molecule described herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby said molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, whereby said cancer may be liver cancer. The molecule for treating liver cancer can be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, said antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, said antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at sites N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0376] In some embodiments, this document provides a method of treating a target cancer by administering a molecule described herein, the molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, whereby the cancer may be head and neck cancer. The molecule for treating head and neck cancer can be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at sites N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0377] In some embodiments, this document provides a method of treating a target cancer by administering a molecule described herein, said molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby said molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, whereby said cancer may be breast cancer. The molecule for treating breast cancer may be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, said antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, said antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at sites N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0378] In some embodiments, this document provides a method for treating a target cancer by administering a molecule described herein, the molecule having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1, whereby the cancer may be gastric cancer. The molecule for treating gastric cancer can be any molecule described herein having an antigen-binding fragment that specifically binds to BTN1A1 or a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen-binding fragment preferentially binds to glycosylated BTN1A1 relative to non-glycosylated BTN1A1. In some embodiments, the antigen-binding fragment preferentially binds to BTN1A1 dimers (e.g., glycosylated BTN1A1 dimers) relative to BTN1A1 monomers (e.g., glycosylated BTN1A1 monomers). In some embodiments, the antigen-binding fragment immune-specifically masks BTN1A1 glycosylation at sites N55, N215, N449, or any combination thereof. In some embodiments, the method further includes administration of anti-PD1 therapy or anti-PD-L1 therapy.

[0379] 5.4.2 Administration Methods This document also provides a method for administering a therapeutically effective amount of the antibodies or molecules provided herein to a patient in need of treatment using an antigen-binding fragment having an immune-specific binding fragment to BTN1A1 or a BTN1A1 ligand, an anti-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) antibody, or other molecules as an antitumor agent. In some embodiments, the patient is a cancer patient.

[0380] Various delivery systems are also known for administering the anti-BTN1A1 antibody or other molecules, or related pharmaceutical compositions, that have an antigen-binding fragment having immune-specific binding to BTN1A1, glycosylated BTN1A1, or a BTN1A1 dimer (e.g., glycosylated BTN1A1 dimer), such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), or as part of a retrovirus or other vector to construct nucleic acid, etc.

[0381] The methods of administration provided herein include, but are not limited to, injection, such as parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered intramuscularly, intravenously, subcutaneously, intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intracavitarily, transepidermally, or epidermally. The compositions can be administered by any convenient route, such as by infusion or bolus injection, absorption through the epithelial or mucosal skin layer (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local. Additionally, pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and formulations with nebulizing agents. See, for example, U.S. Patent Nos. 6,019,968; 5,985,20; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publications Nos. WO92 / 19244; WO97 / 32572; WO97 / 44013; WO98 / 31346; and WO99 / 66903; all of which are incorporated herein by reference in their entirety. In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are applied locally to the area requiring treatment, which can be achieved, for example, by local infusion, by injection, or by an implant, said implant being a porous, non-porous, or gel-like material, including membranes, such as elastic membranes or fibers. In some implementations, care is taken to use materials that are not absorbed by the antibodies or other molecules described herein when administering the antibodies or other molecules described herein.

[0382] In some embodiments, the humanized or chimeric antibodies provided herein are formulated in liposomes for targeted delivery. Liposomes are vesicles containing a concentric phospholipid bilayer encapsulated in an aqueous phase. Liposomes generally contain various types of lipids, phospholipids, and / or surfactants. The components of liposomes are typically arranged in a bilayer structure, similar to the lipid distribution of biological membranes. Liposomes can be useful delivery vectors, partly due to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and provided herein; see, for example, pstein. et al 1985, Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al 1980 Proc. Natl. Acad. Sci. USA,77: 4030-4; U.S. Patent Nos. 4,485,045 and 544,545; all of which are incorporated herein by reference in their entirety.

[0383] This document also provides methods for preparing liposomes with prolonged serum half-life, i.e., enhanced circulation time, such as those disclosed in U.S. Patent No. 5,013,556. In some embodiments, the liposomes used in the methods provided herein are not rapidly cleared from circulation, i.e., are not taken up by the mononuclear phagocyte system (MPS). This document also provides spatially stable liposomes prepared using common methods known to those skilled in the art. Spatially stable liposomes may contain a lipid component with a large and highly flexible hydrophilic portion, which reduces undesirable reactions between the liposome and serum proteins, reduces opsonization with serum components, and reduces MPS recognition. Spatially stable liposomes can be prepared using polyethylene glycol. For the preparation of liposomes and spatially stable liposomes, see, for example, Bendas. et al 2001 BioDrugs, 15(4): 215-224; Allen et al 1987 FEBS Lett. 223: 42-6; Klibanov et al, 1990 FEBSLett, 268: 235-7; Blum et al, 1990, Biochim. Biophys. Acta. , 1029: 91-7; Torchilin et al, 1996, J. LiposomeRes. 6: 99-116; Litzinger et al. , 1994, Biochim. Biophys. Acta, 1190: 99-107; Maruyama et al, 1991, Chem. Pharm. Bull, 39: 1620-2; Klibanov et al, 1991, BiochimBiophysActa, 1062; 142-8; Allen et al, 1994, Adv. DrugDeliv. Rev, 13: 285-309, which are incorporated herein by reference in their entirety.

[0384] This document also provides liposomes suitable for targeting specific organs, see, for example, U.S. Patent No. 4,544,545, or suitable for targeting specific cells, see, for example, U.S. Patent Publication No. 2005 / 0074403, all of which are incorporated herein by reference in their entirety. Liposomes particularly useful in the compositions and methods provided herein can be produced by reverse-phase evaporation using lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes with a desired diameter can be produced by extruding liposomes using a filter material with a specified pore size. In some embodiments, molecules having antigen-binding fragments, such as F(ab'), can be conjugated to liposomes using methods previously described, for example, Martin... et al, 1982, J. Biol. Chem. 257: 286-288, which are incorporated herein by reference in their entirety.

[0385] The humanized or chimeric antibodies described herein can also be formulated as immunoliposomes. An immunoliposome is a liposomal composition in which an antibody or a fragment thereof is covalently or non-covalently attached to the surface of the liposome. The chemistry for attaching an antibody to the surface of a liposome is known in the art; see, for example, U.S. Patent No. 6,787,153; Allen et al, 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44; Hansen et al, 1995, Biochim. Biophys. Acta, 1239: 133-144, which are incorporated herein by reference in their entirety. In some embodiments, the immunoliposomes used in the methods and compositions provided herein are further spatially stabilized. In some embodiments, the humanized antibodies described herein are covalently or non-covalently linked to a hydrophobic anchor that is stably embedded in the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, such as phosphatidylethanolamine (PE) and phosphatidylinositol (PI). To achieve the covalent link between the antibody and the hydrophobic anchor, any biochemical strategy known in the art can be used; see, for example, J. Thomas August ed., 1997. Gene Therapy: Advances in Pharmacology Volume 40, Academic Press, San Diego, California, pp. 399-435, incorporated herein by reference in their entirety. For example, functional groups on antibody molecules can react with active groups on hydrophobic anchors associated with liposomes; for instance, the amino group of the lysine side chain on an antibody can be coupled with liposome-associated N-glutarylphosphatidylethanolamine activated by a water-sol...

Claims

1. A molecule comprising an antigen binding fragment that immunospecifically binds to BTN1A1, wherein the molecule inhibits the binding of BTN1A1 to a BTN1A1 ligand selected from the group consisting of galectin-1 (GAL-1), galectin-9 (GAL-9), NRP-2 (NrP-2), and B- and T-lymphocyte attenuator protein (BTLA).

2. The molecule of claim 1, wherein the antigen binding fragment immunospecifically binds to BTN1A1 and the molecule inhibits the binding of BTN1A1 to GAL-1.

3. The molecule of claim 1 or 2, wherein the antigen binding fragment immunospecifically binds to BTN1A1 and the molecule inhibits the binding of BTN1A1 to GAL-9.

4. The molecule of any one of claims 1-3, wherein the antigen binding fragment immunospecifically binds to BTN1A1 and the molecule inhibits the binding of BTN1A1 to NRP-2.

5. The molecule of any one of claims 1-4, wherein the antigen binding fragment immunospecifically binds to BTN1A1 and the molecule inhibits the binding of BTN1A1 to BTLA.

6. The molecule of any one of claims 1-5, wherein the antigen binding fragment immunospecifically binds to BTN1A1 and the molecule inhibits the binding of BTN1A1 to two or more BTN1A1 ligands selected from the group consisting of GAL-1, GAL-9, NRP-2, or BTLA.

7. The molecule of any one of claims 1-7, wherein the antigen binding fragment immunospecifically binds to the extracellular domain (ECD) of BTN1A1.

8. A molecule comprising an antigen binding fragment that immunospecifically binds to a BTN1A1 ligand selected from the group consisting of GAL-1, GAL-9, NRP-2, and BTLA, wherein the molecule inhibits the binding of the BTN1A1 ligand to BTN1A1.

9. The molecule of claim 8, wherein the antigen binding fragment immunospecifically binds to GAL-1 and the molecule inhibits the binding of GAL-1 to BTN1A1.

10. The molecule of claim 8, wherein the antigen binding fragment immunospecifically binds to GAL-9 and the molecule inhibits the binding of GAL-9 to BTN1A1.

Images