Combination therapies against cancer targeting CD38 and TGF-beta

Abstract

To provide combination cancer therapies.SOLUTION: The present disclosure relates to combination cancer therapies targeting CD38 and TGF-β using antibodies specific for these targets. Also provided are compositions useful in the therapies.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 696,198, filed July 10, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The electronic copy of the Sequence Listing was created on July 9, 2019, is named 022548_WO025_SL.txt, and is 52,428 bytes in size. [Background technology]

[0003] Transforming growth factor beta (TGF-β) is a cytokine that regulates diverse biological processes, such as extracellular matrix formation, wound healing, embryonic development, bone development, hematopoiesis, immune and inflammatory responses, and malignant transformation. Deregulation of TGF-β leads to pathological conditions, such as birth defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

[0004] TGF-β has three known isoforms: TGF-β1, 2, and 3. The three isoforms are pleiotropic in their functions and are expressed in different patterns across cell and tissue types. Although they have similar in vitro activities, knockout of each isoform in a specific cell type suggests that their in vivo roles are not identical, despite their shared ability to bind to the same receptor (Non-Patent Document 1).

[0005] Upon binding of TGF-β to TGFβRII, the receptor's constitutive kinase activity phosphorylates and activates TGFβRI, which phosphorylates SMAD2 / 3, allowing it to interact with SMAD4, which then localizes to the nucleus and initiates transcription of TGF-β-responsive genes (Id.). In addition to this canonical signaling cascade, non-canonical pathways transmit signals through other factors, including p38 MAPK, PI3K, AKT, JUN, JNK, and NF-κB. TGF-β signaling is also regulated by other pathways, including WNT, Hedgehog, Notch, INF, TNF, and RAS. Thus, the ultimate outcome of TGF-β signaling is crosstalk between all these signaling pathways, which integrates the cellular state and environment (Id.).

[0006] CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional ectoenzyme and NAD + It catalyzes the conversion of CD38 to cyclic ADP-ribose (cADPR) and hydrolyzes cADPR to ADP-ribose. During ontogeny, CD38 appears on CD34-positive committed stem cells and lineage-committed progenitors of lymphoid, erythroid, and myeloid cells. CD38 expression persists at different stages of T and B cell development, mostly in the lymphoid lineage, with varying expression levels.

[0007] CD38 is expressed on many hematopoietic malignancies and cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), B- and T-acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), and hairy cell leukemia (HCL). CD38 is upregulated in hematopoietic tumors (including hematopoietic leukemia (HML) and Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). On the other hand, the most primitive pluripotent stem cells of the hematopoietic system are CD38 negative. The correlation of CD38 expression with disease progression in hematopoietic malignancies makes CD38 an attractive target for antibody therapy. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Akhurst et al., Nat Rev Drug Discov 11(10):790~811 (2012) Summary of the Invention [Means for solving the problem]

[0009] Provided herein is a combination therapy that targets CD38 and TGF-β.The inventors have found that anti-TGF-β antibody blocks the ability of TGF-β to dampen the anti-tumor effect of anti-CD38 antibody (for example, TGF-β can inhibit the NK cell-mediated ADCC of anti-CD38 antibody).Compared with currently available treatments for cancer, including antibody treatment, the combination therapy provided herein can provide superior clinical efficacy.

[0010] Thus, provided herein is a method for enhancing the efficacy of an agent that specifically binds CD38 with an agent that specifically binds TGF-β. In some embodiments, provided herein is a method for treating cancer in a patient (e.g., a human patient) by administering to the patient an agent that specifically binds human CD38 and an agent that specifically binds human TGF-β. In some embodiments, the agent that specifically binds human CD38 is an anti-CD38 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-CD38 antibody is capable of killing CD38-positive cells by apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and / or complement-dependent cytotoxicity (CDC), wherein killing CD38-positive cells by apoptosis can occur in the absence of stromal cells or stromal-derived cytokines. In some embodiments, the agent that specifically binds human TGF-β is a pan-specific anti-TGF-β antibody or an antigen-binding fragment thereof.

[0011] In some embodiments, provided herein are methods of treating cancer in a human patient in need thereof, comprising administering to the patient an anti-CD38 antibody and an anti-TGF-β antibody or antigen-fragment thereof.

[0012] In some embodiments, the anti-CD38 antibody is: a) having HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 15 to 20, respectively; b) having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively; or c) has a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0013] In some embodiments, the anti-TGF-β antibody is: a) having HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 5 to 10, respectively; b) having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; or c) has a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2.

[0014] In some embodiments, the anti-CD38 antibody has a heavy chain CDR1 (HCDR1), HCDR2, HCDR3, and a light chain CDR1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 15-20, respectively, and the anti-TGF-β antibody or antigen-binding fragment thereof has a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 5-10, respectively. In certain embodiments, the anti-CD38 antibody has a heavy chain variable domain (V) comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively. H ) and the light chain variable domain (V L ) comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; and the anti-TGF-β antibodies comprise V H and VL In certain embodiments, the anti-CD38 antibody has a heavy chain (HC) and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively; and the anti-TGF-β antibody has a heavy chain and a light chain comprising the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

[0015] In any of the embodiments provided herein, the anti-CD38 antibody may comprise a human IgG1 Fc region, and the anti-TGF-β antibody may comprise a human IgG4 Fc region.

[0016] Also provided herein is a method of treating multiple myeloma in a human patient in need thereof, comprising administering to the patient an anti-CD38 antibody comprising the heavy chain variable domain amino acid sequence of SEQ ID NO: 13 and the light chain variable domain amino acid sequence of SEQ ID NO: 14, and an anti-TGF-β antibody comprising the heavy chain variable domain amino acid sequence of SEQ ID NO: 3 and the light chain variable domain amino acid sequence of SEQ ID NO: 4.

[0017] Also provided herein is a method of treating multiple myeloma in a human patient in need thereof, comprising administering to the patient an anti-CD38 antibody comprising a heavy chain amino acid sequence of SEQ ID NO:11 and a light chain amino acid sequence of SEQ ID NO:12, and an anti-TGF-β antibody comprising a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:2.

[0018] In any of the embodiments provided herein, the anti-CD38 antibody and the anti-TGF-β antibody or fragment are administered sequentially to the patient.

[0019] Also provided herein are anti-CD38 antibodies in combination with anti-TGF-β antibodies for use in treating cancer in a human patient in need thereof, and uses of anti-CD38 antibodies in combination with anti-TGF-β antibodies for the manufacture of a medicament for treating cancer in a human patient in need thereof. In some embodiments, the anti-CD38 antibody a) having HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 15 to 20, respectively; b) having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively; or c) has a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0020] Also provided herein are anti-TGF-β antibodies in combination with anti-CD38 antibodies for use in treating cancer in a human patient in need thereof, and uses of anti-TGF-β antibodies in combination with anti-CD38 antibodies for the manufacture of a medicament for treating cancer in a human patient in need thereof. In some embodiments, the anti-TGF-β antibody a) having HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 5 to 10, respectively; b) having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; or c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2 Has.

[0021] In some embodiments of the methods, antibodies for use, and uses of antibodies described herein, the cancer is CD38 positive.

[0022] In some embodiments of the methods, antibodies for uses, and uses of antibodies described herein, the cancer is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, melanoma, glioblastoma, lung cancer, cutaneous squamous cell carcinoma, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma.

[0023] In some embodiments of the methods, antibodies for use, and uses of antibodies described herein, the cancer is a hematological malignancy.

[0024] In some embodiments of the methods, antibodies for use, and uses of antibodies described herein, the cancer is multiple myeloma. In certain embodiments, the treatment described herein results in less bone destruction and / or enhances bone formation, resulting in bone remodeling and / or fracture healing in multiple myeloma patients than treatment with an anti-CD38 antibody alone.

[0025] Thus, in some embodiments, provided herein are methods for reducing bone destruction in human multiple myeloma patients, comprising administering to the patient an agent that specifically binds human CD38 (e.g., an anti-CD38 antibody) described herein and an agent that specifically binds human TGF-β (e.g., an anti-TGF-β antibody) described herein. Also provided herein are anti-TGF-β antibodies described herein in combination with anti-CD38 antibodies for use in reducing bone destruction in human multiple myeloma patients, and the use of anti-TGF-β antibodies in combination with anti-CD38 antibodies for the manufacture of a medicament for reducing bone destruction in human multiple myeloma patients.

[0026] Also provided herein are methods for enhancing bone formation, which results in bone remodeling and / or fracture healing, in multiple myeloma patients, comprising administering to the patient an agent that specifically binds human CD38 (e.g., an anti-CD38 antibody) described herein and an agent that specifically binds human TGF-β (e.g., an anti-TGF-β antibody) described herein. Also provided herein are anti-TGF-β antibodies described herein in combination with anti-CD38 antibodies for use in enhancing bone formation in human multiple myeloma patients, and the use of anti-TGF-β antibodies in combination with anti-CD38 antibodies for the manufacture of a medicament for enhancing bone formation in human multiple myeloma patients.

[0027] In some embodiments of the methods, antibodies for use, and uses of antibodies described herein, the cancer is refractory to treatment with Ab2, daratumumab, or both. In some embodiments, the patient has relapsed or refractory multiple myeloma and has received at least one prior therapy or at least two prior therapies.

[0028] Also provided herein are articles of manufacture comprising an anti-CD38 antibody and an anti-TGF-β antibody, wherein the article is suitable for treating cancer in a patient, e.g., for the treatment methods described herein. In some embodiments, the anti-CD38 antibody is a) having HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 15 to 20, respectively; b) having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively; or c) having a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 12; Anti-TGF-β antibodies a) having HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 5 to 10, respectively; b) having a heavy chain variable domain and a light chain variable domain comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; or c) has a heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a light chain comprising the amino acid sequence of SEQ ID NO:2. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 shows a graph depicting the release of TGF-β by multiple myeloma cell lines over 1, 2, or 3 days as quantified by ELISA. [Figure 2]FIG. 10 is a graph showing calcein fluorescence levels (% lysis) from MOLP8 target cells incubated with Ab2 at concentrations of 0, 0.001, 0.1, or 1 μg / mL and lysed for 1 hour by human NK cells incubated with 10 ng / mL TGF-β overnight, 24 hours, 48 ​​hours, or 72 hours. [Figure 3] FIG. 10 shows a graph depicting calcein fluorescence levels (% lysis) from MOLP8 target cells incubated with Ab2 at concentrations of 0, 0.001, 0.01, 0.1, or 1 μg / mL or IgG1 control and lysed for 1 hour by human NK cells incubated with 10 ng / mL TGF-β and 50 μg / mL Ab1 for 90 hours. [Figure 4] FIG. 1 is a graph showing the effect of 0.1, 1, and 10 ng / mL TGF-β on Ab2-mediated ADCC of NCI-H929 cells. [Figure 5] Figure 1 shows a graph showing Ab2-mediated ADCC of NCI-H929 cells treated with 100 μg / mL IgG4, 100 μg / mL Ab1, 10 ng / mL TGF-β, TGF-β+IgG4, or TGF-β+Ab1. *=p<0.05 vs. NT; **=p<0.005 vs. NT. [Figure 6] Figure 1 shows a graph showing Ab2-mediated ADCC of NCI-H929 cells treated with 200 μg / mL IgG4, 200 μg / mL Ab1, 10 ng / mL TGF-β, TGF-β+IgG4, or TGF-β+Ab1. *=p<0.05 vs. NT; **=p<0.005 vs. NT. [Figure 7] FIG. 10 is a graph showing calcein fluorescence levels (% lysis) from K562 target cells lysed by the cytolytic activity of endogenous TGF-β-releasing JJN3 cells and human NK cells incubated with 100 μg / mL Ab1 or isotype control for 90 hours. [Figure 8]FIG. 16 shows a graph showing Ab2-mediated ADCC of RPMI8226 target cells incubated with Ab2 at concentrations of 0, 0.01, or 0.1 μg / mL or control Ab2 variants at a concentration of 0.1 μg / mL and lysed for 1 hour by endogenous TGF-β-releasing JJN3 cells and human NK cells incubated with 100 μg / mL Ab1 or isotype control for 90 hours. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure provides new combination therapies that target human TGF-β and human CD38 (e.g., by using antibodies that bind to these targets). The combination therapies are used to treat conditions such as cancer. Unless otherwise specified, "TGF-β" refers herein to human TGF-β. The polypeptide sequences of the three isoforms of human TGF-β (TGF-β1, TGF-β2, and TGF-β3) are available under SwissProt accession numbers P01137, P08112, and P10600, respectively. and are represented herein as SEQ ID NOS: 21-23. Unless otherwise specified, "CD38" refers herein to human CD38. The human CD38 polypeptide sequence is available under Genbank accession number NP_001766 and is represented herein as SEQ ID NO: 24.

[0031] As used herein, the term "antibody (Ab)" or "immunoglobulin (Ig)" refers to a tetrameric protein containing two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa) interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and heavy chain constant domain (C H Each light chain is composed of a light chain variable region (V L ) and the light chain constant region (C L ) V H and V LThe domains are further subdivided into regions of hypervariability, called "complementarity-determining regions (CDRs)", interspersed with regions that are more conserved, called "framework regions (FRs)". H or V L is composed of three CDRs and four FRs arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each region are based on the IMGT® definition (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or Kabat, Sequences of Proteins of The definition may be as defined by the Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989).

[0032] The term "affinity" refers to a measure of the attractive force between an antigen and an antibody. The intrinsic attractiveness of an antibody for an antigen is determined by the binding affinity equilibrium constant (K D ) is typically expressed as K D 10 as determined, for example, by surface plasmon resonance or Bio-Layer Interferometry -7 Less than M, e.g., 10 -8 When the binding affinity is less than M (for example, 1 to 9 nM), the antibody can be said to specifically bind to the antigen.

[0033] The term "k" off " refers to the dissociation rate constant of a particular antibody-antigen interaction. off The dissociation rate constant is measured, for example, by Bio-Layer Interferometry.

[0034] As used herein, the term "epitope" refers to the portion (determinant) of an antigen that specifically binds to an antibody. Epitope determinants generally consist of chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and generally have specific three-dimensional structural and charge characteristics. Epitopes can be "linear" or "conformational." In a linear epitope, all points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues of the protein that are separated from each other in the primary amino acid sequence. Once a desired epitope on an antigen has been determined, antibodies against that epitope can be generated using techniques well known in the art. For example, antibodies against a linear epitope can be generated by immunizing an animal with a peptide containing the amino acid residues of the linear epitope. Antibodies to conformational epitopes are generated, for example, by immunizing an animal with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies to specific epitopes can also be generated, for example, by immunizing an animal with the target molecule of interest or a relevant portion thereof, and then screening for epitope binding.

[0035] Whether an antibody binds to the same epitope as an antibody described herein or competes for binding can be determined using methods known in the art, including, but not limited to, competition assays, epitope binning, and alanine scanning. In some embodiments, the test antibody and an antibody described herein bind to at least one common residue (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues) in the target protein (i.e., TGF-β or CD38). In further embodiments, the contact residues in the target protein are completely identical between the test antibody and the antibody described herein. In one embodiment, the antibody described herein is allowed to bind to the target protein under saturating conditions, and then the ability of the test antibody to bind to the target protein can be measured. If the test antibody can bind to the target protein simultaneously with the reference antibody, the test antibody binds to a different epitope from the reference antibody. However, if the test antibody cannot bind to the target protein simultaneously, the test antibody binds to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the antibody described herein. This experiment can be performed using, for example, ELISA, RIA, BIACORE TM , SPR, Bio-Layer Interferometry, or flow cytometry. The above competition method may also be used in two directions to assess whether an antibody cross-competes with another antibody (i.e., to determine if a known antibody blocks a test antibody, and vice versa).

[0036] The term "humanized" refers to the fact that when an antibody is of wholly or partially non-human origin (e.g., a murine antibody obtained by immunizing a mouse with an antigen of interest, or a chimeric antibody based on such a murine antibody), certain amino acids can be replaced, particularly in the framework and constant regions of the heavy and light chains, to avoid or minimize immune responses in humans. Although it is not possible to accurately predict the immunogenicity and therefore the human anti-antibody response of a particular antibody, non-human antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies, in which foreign (e.g., rodent) constant regions are replaced with sequences of human origin, have been shown to be generally less immunogenic than antibodies of completely foreign origin, and the trend in therapeutic antibodies is toward humanized or fully human antibodies. Therefore, chimeric or other antibodies of non-human origin are humanized to reduce the risk of human anti-antibody responses.

[0037] For chimeric antibodies, humanization typically involves modifying the framework regions of the variable domain sequences. While amino acid residues that are part of the complementarity-determining regions (CDRs) are often not altered in connection with humanization, in certain cases it may be desirable to alter individual CDR amino acid residues, for example, to remove glycosylation sites, deamidation sites, aspartic acid isomerization sites, or undesired cysteine ​​or methionine residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr (where X is any amino acid except Pro). Removal of N-glycosylation sites is achieved by mutating the Asn or Ser / Thr residue to a different residue, preferably by means of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when they occur in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences (e.g., Asn-Ala). Thus, when such a deamidation site, particularly Asn-Gly, occurs in a CDR sequence, it would be desirable to remove the site, typically by removing one of the residues related by a conservative substitution.

[0038] Numerous methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson, Front Biosci. 13:1619-1633 (2008). One commonly used method is to humanize CDRs. Grafting, for example, involves identifying human germline gene counterparts for mouse variable domain genes and grafting mouse CDR sequences onto this framework for mouse-derived chimeric antibodies. The specificity of an antibody's interaction with a target antigen is primarily vested in the amino acid residues located in the six CDRs of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are more variable between individual antibodies than the sequences outside the CDRs. Because the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody, or more generally, any particular antibody with a given amino acid sequence, for example, by constructing an expression vector that expresses CDR sequences from a particular antibody grafted onto framework sequences from a different antibody. As a result, it is possible to "humanize" a non-human antibody and still substantially maintain the binding specificity and affinity of the original antibody. CDR grafting may be based on the Kabat CDR definitions, although more recent publications (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007)) suggest that the IMGT® definitions (the international ImMunoGeneTics information system®, www.imgt.org) may improve humanization results (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)).

[0039] In some cases, CDR grafting may reduce the binding specificity and affinity, and therefore the biological activity, of the CDR-grafted non-human antibody compared to the parent antibody from which the CDRs are obtained. Back mutations (sometimes referred to as "framework repairs") are introduced into selected positions (typically framework regions) of the CDR-grafted antibody to re-establish the binding specificity and affinity of the parent antibody. Positions for possible back mutations are identified using information available in the literature and antibody databases. Candidate amino acid residues for back mutations are typically those located on the surface of the antibody molecule, while buried residues with low surface exposure are usually not altered.

[0040] An alternative humanization technique to CDR grafting and backmutation is resurfacing, in which non-surface exposed residues of the non-human origin are retained, but surface residues are changed to human residues.

[0041] In certain cases, it is desirable to alter one or more CDR amino acid residues to improve binding affinity for the target epitope. This is known as "affinity maturation." Various affinity maturation methods are known in the art, see, for example, Burks et al. et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method of Wu et al., Proc Natl Acad Sci USA, 95:6037-6042 (1998).

[0042] The term "human antibody" refers to an antibody in which the variable domain and constant region sequences are derived from human sequences. The term encompasses antibodies that have sequences derived from human genes, but in which those sequences have been modified, for example, to reduce immunogenicity, increase affinity, and increase stability. The term encompasses antibodies that are recombinantly produced in non-human cells, which may provide glycosylation that is not typical of human cells. The term also encompasses antibodies produced in transgenic non-human organisms with human antibody genes.

[0043] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to a portion or fragment of an antibody that retains the ability to specifically bind to an antigen. In some embodiments, an antigen-binding fragment of the present disclosure is a Fab, Fab', F(ab')2, Fv, or scFv fragment. In certain embodiments, an antigen-binding fragment of the present disclosure is F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region (Fab is a V L , V H , C L and C H1 In some embodiments, the antigen-binding fragment of the present disclosure is a monovalent antibody fragment consisting of a C domain. H2 or C H3 In some cases, the fragments may comprise a domain. Antibody fragments are prepared from whole antibodies using conventional techniques, such as, for example, papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody fragments, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, for example, as described herein.

[0044] The antibodies and antigen-binding fragments described herein are isolated. The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, by virtue of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state; (2) is substantially free from other proteins from the same species; (3) is expressed by cells from a different species; or (4) does not occur naturally. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the original cell is "isolated" from its naturally associated components. A protein can also be substantially freed from naturally associated components by isolation using protein purification techniques well known in the art.

[0045] The class (isotype) and subclass of the antibody described herein can be determined by any method known in the art. Generally, the class and subclass of an antibody are determined using antibodies specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blot, and other techniques. Alternatively, the class and subclass can be determined by sequencing all or part of the constant region of the heavy and / or light chain of the antibody, comparing their amino acid sequences with the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibody. The preferred isotype of the present disclosure is the IgG isotype.

[0046] TGF-β inhibitors In some embodiments, the agent that specifically binds TGF-β used in the combination therapies described herein is an anti-TGF-β antibody or antigen-binding fragment thereof (e.g., Ab1, fresolimumab, XOMA 089 / NIS793 (Gramont et al., Oncoimmunology 6(1):e1257453 (2017)), SRK-181 (Scholar Rock Inc.), ABBV-151 (AbbVie Inc.), lerdelimumab, or metelimumab) or a TGF-β trap molecule (e.g., M7824 (Knudson et al., Oncoimmunology 7(5):e1426519 (2018)) or AVID200 (Thwaites et al., Blood 130:2532 (2017))). In certain embodiments, the anti-TGF-β antibody is a human monoclonal antibody. In some embodiments, the anti-TGF-β antibody is a pan-TGF-β specific monoclonal antibody that has a reduced tendency to form half-antibodies compared to known antibodies, such as fresolimumab. In some embodiments, the anti-TGF-β antibody has a superior pharmacokinetic profile (e.g., higher exposure in the body compared to fresolimumab).

[0047] In some embodiments, the anti-TGF-β antibody is an antibody described in PCT Patent Publication No. WO2018 / 134681, PCT Patent Publication No. WO2006 / 086469, PCT Patent Publication No. WO2014 / 153435, U.S. Patent No. 8,569,462, or U.S. Patent No. 7,527,791, which are incorporated by reference in their entireties. In certain embodiments, the anti-TGF-β antibody is antibody Ab1 or a variant thereof. The variants may contain, for example, certain minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may be in, for example, framework regions) compared to Ab1 that do not result in a loss of antigen-binding specificity of the antibody.

[0048] Antibody Ab1 has a predicted molecular weight of 144 kD when unglycosylated. Its heavy and light chain amino acid sequences are SEQ ID NOs: 1 and 2, respectively. These two sequences are shown below. The variable domains are in italics. The CDRs are shown in boxes. The glycosylation site in the constant domain of the heavy chain is in bold and lowercase (N297). Ab1 has a human IgG4 constant region, with residue 228 (EU numbering) in the hinge region mutated from serine to proline. P228 is in bold in a box in the sequence of SEQ ID NO: 1 shown below.

[0049] [ka]

[0050] In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments described herein compete with Ab1 for binding to TGF-β or bind to the same epitope on TGF-β as Ab1.

[0051] In some embodiments, the anti-TGF-β antibodies described herein have a heavy chain comprising: a) heavy chain CDR1-3 (HCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 5-7, respectively; b) a heavy chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 3 H ); c) V comprising the amino acid sequence of SEQ ID NO: 3 H ;or d) The amino acid sequence of SEQ ID NO:1.

[0052] In some embodiments, the anti-TGF-β antibodies described herein have a light chain comprising: a) light chain CDR1-3 (LCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 8 to 10, respectively; b) a light chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO:4 L ); c) V comprising the amino acid sequence of SEQ ID NO: 4 L ;or d) The amino acid sequence of SEQ ID NO:2.

[0053] In some embodiments, the anti-TGF-β antibodies described herein comprise any combination of the heavy chains described above with any of the light chains described above.

[0054] In some embodiments, the anti-TGF-β antibodies described herein include: a) HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 5 to 10, respectively; b) a V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO:3 H and V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO:4. L ; c) V comprising the amino acid sequence of SEQ ID NO: 3 H and V comprising the amino acid sequence of SEQ ID NO:4 L and d) HC comprising the amino acid sequence of SEQ ID NO:1 and LC comprising the amino acid sequence of SEQ ID NO:2.

[0055] In some embodiments, the six CDRs or V of an anti-TGF-β antibody described herein (e.g., Ab1) are H and V LThe anti-TGF-β antibody having the formula (I) has a human IgG4 constant region. In certain embodiments, residue 228 (EU numbering) in the hinge region of the IgG4 constant region is mutated from serine to proline. The constant domain of the anti-TGF-β antibody described herein can also be modified, for example, at Kabat residue L248 (e.g., by introducing the mutation L248E) to reduce any undesired effector functions of the molecule.

[0056] In some embodiments, the anti-TGF-β antibodies described herein have a human immunoglobulin kappa light chain region.

[0057] In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments described herein specifically bind to human TGF-β1, -β2, and -β3.

[0058] In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments described herein are combined with TGF-β1, -β2, and / or -β3 (e.g., TGF-β1, -β2, and -β3) at a concentration of 1×10 -8 M or less, e.g., 9 x 10 -9 M, 8 x 10 -9 M, 7 x 10 -9 M, 6 x 10 -9 M, 5 x 10 -9 M, 4 x 10 -9 M, 3 x 10 -9 M, 2 x 10 -9 M, or 1 x 10 -9 K of M D In certain embodiments, the anti-TGF-β antibody or antigen-binding fragment binds to TGF-β1, -β2, and -β3 at 3×10 -9 M or lower K D Combine with.

[0059] In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments described herein have one or more of the following properties: a) inhibit TGF-β signaling; b) neutralizes TGF-β when assayed in a mink lung epithelial cell assay; c) has an EC50 of about 0.05 to 1 μg / ml as determined in an A549 cell IL-11 induction assay; d) inhibiting the differentiation of CD4+ T cells into inducible regulatory T cells (iTreg); e) reducing the immunosuppressive tumor microenvironment; f) increasing MIP2 levels in a patient (e.g., in the patient's tumor tissue); g) increasing KC / GRO levels in the patient (e.g., in the patient's tumor tissue); h) promoting the activation or infiltration of CD8-positive T cells, for example, INF-γ-positive CD8-positive T cells, into tumor tissue; i) increasing the clustering of natural killer (NK) cells in a patient (e.g., in the patient's tumor tissue); and j) Restoring the cytolytic activity of NK-92 cells after incubation with human recombinant TGF-β. In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments described herein have one, two, three, four, five, six, seven, eight, nine, or all of the above properties.

[0060] In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments described herein have an increased half-life, increased exposure, or both, compared to fresolimumab. For example, the increase is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. The exposure of a drug, such as an antibody or fragment described herein, is a function of the concentration of the drug in the body over time. The concentration of a drug in the body is often indicated by the drug level in blood, plasma, or serum. The half-life and exposure (biological exposure) of a drug are measured by well-known methods (e.g., as described in PCT Patent Publication WO2018 / 134681).

[0061] CD38 inhibitors In some embodiments, the agent that specifically binds to CD38 used in the combination therapy described herein is an anti-CD38 antibody or antigen-binding fragment thereof. In certain embodiments, the anti-CD38 antibody is a humanized monoclonal antibody. In some embodiments, the anti-CD38 antibody is an antibody described in U.S. Patent No. 8,153,765, the entire contents of which are incorporated herein by reference. In some embodiments, the anti-CD38 antibody is an antibody obtained from the American Type Culture Collection under deposit number PTA-7670. The anti-CD38 antibody is produced by a hybridoma cell line deposited in the Collection. In some embodiments, the anti-CD38 antibody is Ab2, Ab3, Ab4, daratumumab, MOR202 (Raab et al., Blood 128:1152 (2016)), TAK-079 (Roepcke et al., Pharmacol Res Perspect 6(3):e00402 (2018)), TAK-573 (Takeda Pharmaceutical Company Limited), TAK-169 (Takeda Pharmaceutical Company Limited), HexaBody®-CD38 (Genmab / Janssen), anti-CD38 SIFbody (Momenta Pharmaceuticals, Inc.), or TSK011010 (CASI Pharmaceuticals Inc.).

[0062] In certain embodiments, the anti-CD38 antibody is antibody Ab2 or a variant thereof, where the variant may contain certain minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may be, for example, in framework regions) compared to Ab2 that do not result in a loss of antigen-binding specificity of the antibody.

[0063] The heavy and light chain amino acid sequences of Ab2 are SEQ ID NOs: 11 and 12, respectively. These two sequences are shown below. The variable domains are in italics. The CDRs are shown in boxes. Ab2 has a human IgG1 constant region.

[0064] [ka]

[0065] In some embodiments, the anti-CD38 antibody or antigen-binding fragment competes with Ab2 for binding to CD38 or binds to the same epitope on CD38 as Ab2.

[0066] In some embodiments, the anti-CD38 antibody has a heavy chain comprising: a) heavy chain CDR1-3 (HCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 15-17, respectively; b) a heavy chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 13 H ); c) V comprising the amino acid sequence of SEQ ID NO: 13 H ;or d) The amino acid sequence of SEQ ID NO: 11.

[0067] In some embodiments, the anti-CD38 antibody has a light chain comprising: a) light chain CDR1-3 (LCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 18 to 20, respectively; b) a light chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 14. L ); c) V comprising the amino acid sequence of SEQ ID NO: 14 L ;or d) The amino acid sequence of SEQ ID NO: 12.

[0068] In some embodiments, the anti-CD38 antibodies described herein comprise any combination of the heavy chains described above with any of the light chains described above.

[0069] In some embodiments, the anti-CD38 antibodies described herein include: a) HCDR1-3 and LCDR1-3, each comprising the amino acid sequences of SEQ ID NOs: 15 to 20; b) a V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 13 H and V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 14. L ; c) V comprising the amino acid sequence of SEQ ID NO: 13 H and V comprising the amino acid sequence of SEQ ID NO: 14 L and d) HC comprising the amino acid sequence of SEQ ID NO: 11 and LC comprising the amino acid sequence of SEQ ID NO: 12.

[0070] In certain embodiments, the anti-CD38 antibody is antibody Ab3 or a variant thereof, where the variant may contain certain minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may be, for example, in framework regions) compared to Ab3 that do not result in a loss of antigen-binding specificity of the antibody.

[0071] The heavy and light chain amino acid sequences of Ab3 are SEQ ID NOs: 25 and 26, respectively. These two sequences are shown below. The variable domains are in italics. The CDRs are shown in boxes.

[0072] [ka]

[0073] In some embodiments, the anti-CD38 antibody or antigen-binding fragment competes with Ab3 for binding to CD38 or binds to the same epitope on CD38 as Ab3.

[0074] In some embodiments, the anti-CD38 antibody has a heavy chain comprising: a) heavy chain CDR1-3 (HCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 29-31, respectively; b) a heavy chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 27 H ); c) V comprising the amino acid sequence of SEQ ID NO: 27 H ;or d) The amino acid sequence of SEQ ID NO: 25.

[0075] In some embodiments, the anti-CD38 antibody has a light chain comprising: a) light chain CDR1-3 (LCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 32 to 34, respectively; b) a light chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 28 L ); c) V comprising the amino acid sequence of SEQ ID NO: 28 L ;or d) The amino acid sequence of SEQ ID NO: 26.

[0076] In some embodiments, the anti-CD38 antibodies described herein comprise any combination of the heavy chains described above with any of the light chains described above.

[0077] In some embodiments, the anti-CD38 antibodies described herein include: a) HCDR1-3 and LCDR1-3, each comprising the amino acid sequences of SEQ ID NOs: 29 to 34; b) a V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 27 H and V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 28. L ; c) V comprising the amino acid sequence of SEQ ID NO: 27 H and V comprising the amino acid sequence of SEQ ID NO: 28 L ; and d) A HC comprising the amino acid sequence of SEQ ID NO: 25 and a LC comprising the amino acid sequence of SEQ ID NO: 26.

[0078] In certain embodiments, the anti-CD38 antibody is antibody Ab4 or a variant thereof, where the variant may contain certain minimal amino acid changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes, which may be, for example, in framework regions) compared to Ab4 that do not result in a loss of antigen-binding specificity of the antibody.

[0079] The heavy and light chain amino acid sequences of Ab4 are SEQ ID NOs: 35 and 36, respectively. These two sequences are shown below. The variable domains are in italics. The CDRs are shown in boxes.

[0080] [ka]

[0081] In some embodiments, the anti-CD38 antibody or antigen-binding fragment competes with Ab4 for binding to CD38 or binds to the same epitope on CD38 as Ab4.

[0082] In some embodiments, the anti-CD38 antibody has a heavy chain comprising: a) heavy chain CDR1-3 (HCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 39-41, respectively; b) at least 75%, 80%, 85% of the amino acid sequence of SEQ ID NO: 37; heavy chain variable domains (V) that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical H ); c) V comprising the amino acid sequence of SEQ ID NO: 37 H ;or d) The amino acid sequence of SEQ ID NO: 35.

[0083] In some embodiments, the anti-CD38 antibody has a light chain comprising: a) light chain CDR1-3 (LCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 42 to 44, respectively; b) a light chain variable domain (V) that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 38. L ); c) V comprising the amino acid sequence of SEQ ID NO: 38 L ;or d) The amino acid sequence of SEQ ID NO: 36.

[0084] In some embodiments, the anti-CD38 antibodies described herein comprise any combination of the heavy chains described above with any of the light chains described above.

[0085] In some embodiments, the anti-CD38 antibodies described herein include: a) HCDR1-3 and LCDR1-3 containing the amino acid sequences of SEQ ID NOs: 39 to 44, respectively; b) a V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 37 Hand V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 38. L ; c) V comprising the amino acid sequence of SEQ ID NO: 37 H and V comprising the amino acid sequence of SEQ ID NO: 38 L ; and d) A HC comprising the amino acid sequence of SEQ ID NO: 35 and a LC comprising the amino acid sequence of SEQ ID NO: 36.

[0086] In some embodiments, the anti-CD38 antibodies described herein have a human IgG1 constant region.

[0087] In some embodiments, the anti-CD38 antibodies described herein have a human immunoglobulin kappa light chain region.

[0088] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are administered in a concentration of 1×10 -8 M or less, e.g., 9 x 10 -9 M, 8 x 10 -9 M, 7 x 10 -9 M, 6 x 10 -9 M, 5 x 10 -9 M, 4 x 10 -9 M, 3 x 10 -9 M, 2 x 10 -9 M, or 1 x 10 -9 K of M D In certain embodiments, the anti-CD38 antibody or antigen-binding fragment binds to CD38 at a concentration of 3×10 -9 M or lower K D Combine with.

[0089] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein kill CD38-positive cells by apoptosis, ADCC, and CDC; in certain embodiments, killing the CD38-positive cells by apoptosis can occur in the absence of stromal cells or stromal-derived cytokines. In some embodiments, the CD38-positive cells are malignant cells. In some embodiments, the CD38-positive cells are B cells. In certain embodiments, the CD38-positive cells are tumor cells derived from hematopoietic malignancies. In more preferred embodiments, the CD38-positive cells are lymphoma cells, In a more preferred embodiment, the CD38-positive cells are leukemia cells or multiple myeloma cells. In a more preferred embodiment, the CD38-positive cells are NHL, BL, MM, B-CLL, ALL, TCL, AML, HCL, HL, or CML cells.

[0090] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, or 40% (e.g., at least 24%) of Daudi lymphoma cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0091] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (e.g., at least 7%) of Ramos lymphoma cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0092] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (e.g., at least 11%) of MOLP-8 multiple myeloma cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0093] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, or 50% (e.g., at least 36%) of SU-DHL-8 lymphoma cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0094] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 20%, 25%, 26%, 27%, 28%, 29%, 30%, or 35% (e.g., at least 27%) of NU-DUL-1 lymphoma cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0095] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, or 75% (e.g., at least 62%) of DND-41 leukemia cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0096] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or 13% (e.g., at least 9%) of JVM-13 leukemia cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0097] In some embodiments, the anti-CD38 antibodies or antigen-binding fragments described herein are capable of killing at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8% (e.g., at least 4%) of HC-1 leukemia cells in vitro in the absence of stromal cells or stromal-derived cytokines.

[0098] In some embodiments, the agent that specifically binds to CD38 is a conjugate comprising an anti-CD38 antibody or antigen-binding fragment described herein linked to a cytotoxic agent. The cytotoxic agent can be, for example, a maytansinoid, a small drug, a tomaymycin derivative, a repellent, or the like. The cytotoxic agent is selected from tomycin derivatives, prodrugs, taxoids, CC-1065 and CC-1065 analogs, or any of the cytotoxic agents described in U.S. Pat. No. 8,153,765, which is incorporated herein by reference in its entirety.

[0099] The class of the anti-TGF-β or anti-CD38 antibodies described herein is changed or switched to another class or subclass. L or V H The nucleic acid molecule encoding C L or C H The nucleic acid sequence encoding V is isolated using methods well known in the art. L or V HThe nucleic acid molecule encoding the antibody is then operably linked to a nucleic acid sequence encoding a CL or CH chain, respectively, from an immunoglobulin molecule of a different class. This is accomplished using a vector or nucleic acid molecule containing the CL or CH chain, as described above. For example, an antibody that was originally IgM is class switched to IgG. Furthermore, class switching can be used to convert one IgG subclass to another (e.g., from IgG1 to IgG2). The κ light chain constant region is changed to a λ light chain constant region, etc. A preferred method for producing an antibody described herein having a desired Ig isotype includes the steps of isolating a nucleic acid molecule encoding the antibody heavy chain and a nucleic acid molecule encoding the antibody light chain, obtaining the variable domain of the heavy chain, ligating the variable domain of the heavy chain to the constant region of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in cells, and harvesting an antibody having the desired isotype.

[0100] The antibodies described herein may be IgG, IgM, IgE, IgA, or IgD molecules, but are typically of the IgG isotype, e.g., IgG subclass IgG1, IgG 2a or IgG 2b , IgG3, or IgG4.

[0101] In one embodiment, the antibody may contain at least one mutation in the Fc region. Several different Fc mutations are known, and these mutations provide altered effector function. For example, in many cases, it is desirable to reduce or eliminate effector function, such as when ligand / receptor interactions are undesirable or in the case of antibody-drug conjugates.

[0102] In some embodiments, the antibodies described herein, e.g., anti-TGF-β and anti-CD38 antibodies, do not have a C-terminal lysine in their heavy chains. The C-terminal lysine is removed during manufacturing or by recombinant techniques (i.e., the coding sequence for the heavy chain does not contain a codon for the C-terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO: 1 or 11 without the C-terminal lysine are also intended to be within the present disclosure.

[0103] Combination Therapy The present disclosure provides combination therapies comprising an agent that specifically binds human TGF-β and an agent that specifically binds human CD38. In some embodiments, the agent that specifically binds TGF-β is any anti-TGF-β antibody or antigen-binding fragment thereof described herein. In some embodiments, the agent that specifically binds CD38 is any anti-CD38 antibody or antigen-binding fragment thereof described herein. The present disclosure also contemplates combination therapies of one or more other agents that inhibit TGF-β (e.g., galunisertib, LY3200882, PF-06952229 (Pfizer Inc.), GFH-018 (GenFleet Therapeutics Inc.), and / or bactosertib) and / or one or more other agents that inhibit CD38. The combination therapies described herein may take the form of methods of treatment using the agents or pharmaceutical compositions comprising the agents.

[0104] The present disclosure provides antibodies against human CD38 (e.g., anti-CD38 antibodies or antigen-binding fragments thereof, Also contemplated are combination therapies that include an agent that specifically binds to an LAP (eg, those described herein) and an agent that specifically binds to an LAP (eg, an anti-LAP antibody).

[0105] In some embodiments, the combination therapy of the present disclosure uses an anti-TGF-β antibody Ab1 and an anti-CD38 antibody Ab2. In some embodiments, the combination therapy of the present disclosure uses an antibody or antigen-binding fragment thereof that competes with or binds to the same epitope of TGF-β as Ab1 and an antibody that competes with or binds to the same epitope of CD38 as Ab2. In certain embodiments, the Ab1 and Ab2 agents are used in separate compositions (e.g., administered sequentially). In certain embodiments, the Ab1 and Ab2 agents are used in a single composition.

[0106] In certain embodiments, the combination therapy of the present disclosure uses: - an anti-TGF-β antibody or an antigen-binding fragment thereof comprising HCDR1-3 and LCDR1-3 having the amino acid sequences of SEQ ID NOs: 5 to 10, respectively; and an anti-CD38 antibody comprising HCDR1-3 and LCDR1-3 having the amino acid sequences of SEQ ID NOs: 15 to 20, respectively; - V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 3. H and V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO:4. L an anti-TGF-β antibody or antigen-binding fragment thereof comprising: H and V that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to the amino acid sequence of SEQ ID NO: 14. L V having the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; H and V Lan anti-TGF-β antibody or antigen-binding fragment thereof comprising: H and V L an anti-CD38 antibody; or - an anti-TGF-β antibody comprising an HC and an LC having the amino acid sequences of SEQ ID NOs: 1 and 2, respectively; and an anti-CD38 antibody comprising an HC and an LC having the amino acid sequences of SEQ ID NOs: 11 and 12, respectively.

[0107] In some embodiments, the anti-TGF-β and anti-CD38 antibodies used in the combination therapy have the isotypes IgG4 and IgG1, respectively. In certain embodiments, the anti-TGF-β antibody has a human IgG4 constant region, and residue 228 (EU numbering) in the hinge region is mutated from serine to proline.

[0108] In some embodiments, the anti-TGF-β antibodies or antigen-binding fragments thereof described herein ameliorate the immunosuppressive effects of TGF-β in NK cell-mediated ADCC by the anti-CD38 antibodies described herein, making the combination therapy of the present disclosure more effective than treatment with the anti-CD38 antibody alone.

[0109] In some embodiments, a combination therapy comprising an agent that specifically binds human TGF-β and an agent that specifically binds human CD38 (e.g., an anti-TGF-β antibody and an anti-CD38 antibody, e.g., Ab1 and Ab2, respectively) can further comprise an additional agent or therapy. In certain embodiments, the additional agent or therapy is, for example, lenalidomide, pomalidomide, bortezomib, methylprednisolone, dexamethasone, prednisone, melphalan, ixazomib, carfilzomib, thalidomide, cyclophosphamide, pembrolizumab, a pan-histone deacetylase inhibitor (e.g., panobinostat), retinoids, or the like. In certain embodiments, the additional agent or therapy is a combination of lenalidomide and dexamethasone. In certain embodiments, the additional agent or therapy is a combination of bortezomib, melphalan, and prednisone. In certain embodiments, the additional agent or therapy is a combination of bortezomib and dexamethasone. In certain embodiments, the additional agent or therapy is a combination of pomalidomide and dexamethasone. In certain embodiments, the additional agent or therapy is dexamethasone. In some embodiments, the additional agent or therapy is a treatment for a condition targeted by a combination therapy of the present disclosure. For example, if the condition is myeloma (e.g., multiple myeloma), the treatment is, for example, elotuzumab.

[0110] In some embodiments, the agents in the combination therapy of the present disclosure are administered in more than one composition. In certain embodiments, each agent is provided in a separate composition. In cases where more than one composition is present, the compositions are administered simultaneously, sequentially, or separately. In other embodiments, the agents are administered in a single composition. For example, a combination therapy including an anti-TGF-β antibody and an anti-CD38 antibody may involve the administration of a single composition containing both antibodies or separate compositions for each antibody (the separate compositions are administered sequentially or together).

[0111] Therapeutic Uses of the Combination Therapies of the Present Disclosure In one aspect, the combination therapy of the present disclosure is used to treat conditions that depend on CD38 expression.In some embodiments, the combination therapy of the present disclosure is used to treat hyperproliferative diseases, inflammatory diseases, autoimmune diseases, or fibrotic conditions.In certain embodiments, the combination therapy of the present disclosure is used to treat cancer.

[0112] In some embodiments, the combination therapy of the present disclosure targets CD38-positive cells (e.g., CD38-positive cancer cells, e.g., malignant B cells). The cells are identified as CD38-positive by any suitable method of determining gene or protein expression, for example, by histology, flow cytometry, RT-PCR, or RNA-Seq. The cancer cells used for determination are obtained through tumor biopsy or collection of circulating tumor cells. Without wishing to be bound by theory, it is contemplated that an agent that specifically binds to CD38 binds to CD38-positive cells and mediates ADCC / CDC in the cells, and that an agent that specifically binds to TGF-β reduces the immunosuppressive effect of TGF-β, thus enhancing the efficacy of cancer therapy.

[0113] In some embodiments, the combination therapy of the present disclosure is used to treat myeloma, e.g., multiple myeloma (e.g., relapsed and / or refractory multiple myeloma, newly diagnosed multiple myeloma (optionally ineligible for transplant), smoldering multiple myeloma, light chain myeloma, non-secretory myeloma, immunoglobulin D myeloma, or immunoglobulin E myeloma). In certain embodiments, the combination therapy of the present disclosure results in less bone destruction in myeloma bone lesions than therapy with only an agent that specifically binds to CD38 (e.g., an anti-CD38 antibody). In certain embodiments, the combination therapy of the present disclosure may result in improved healing of myeloma bone lesions. Without wishing to be bound by theory, it is contemplated that an agent that specifically binds to TGF-β (e.g., an anti-TGF-β antibody) inhibits the inhibitory activity of TGF-beta on osteoblast differentiation and matrix mineralization, thus enhancing bone formation and resulting in bone remodeling and fracture healing. Furthermore, because mature osteoblasts enhance apoptosis and cell cycle arrest in multiple myeloma cells, agents that specifically bind to TGF-β may also suppress multiple myeloma cell proliferation.

[0114] In some embodiments, the combination therapy of the present disclosure is used to treat, for example, amyloidosis (e.g., recurrent amyloidosis). It is used to treat acute or refractory primary amyloidosis or light-chain amyloidosis), myelodysplastic syndrome (MDS), monoclonal gammopathy, solitary plasmacytoma, extramedullary plasmacytoma, or Waldenstrom's macroglobulinemia.

[0115] In some embodiments, the combination therapy of the present disclosure is used to treat a hematological malignancy, such as leukemia or lymphoma. For example, the malignant tumor is chronic lymphocytic leukemia, B and T acute lymphocytic leukemia, acute lymphoblastic leukemia (e.g., B-cell acute lymphoblastic leukemia or B-cell or T-cell precursor acute lymphoblastic leukemia), chronic lymphocytic acute myeloid leukemia, chronic myelogenous leukemia, acute myeloid leukemia, chronic myeloid leukemia, promyelocytic leukemia, and hairy cell leukemia), non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma or germinal center B-cell lymphoma), T-cell lymphoma (e.g., peripheral T-cell lymphoma), natural killer / T-cell lymphoma (e.g., nasal type), lymphoblastic lymphoma, mantle cell lymphoma, and follicular lymphoma. In certain embodiments, the combination therapy of the present disclosure is used to treat lymphoblastic leukemia.

[0116] Other cancers that may be treated by the combination therapy of the present disclosure are solid tumors, including, but not limited to, skin cancer (e.g., melanoma, unresectable or metastatic melanoma, cutaneous squamous cell carcinoma, xeroderma pigmentosum, and keratoacanthoma), thyroid cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, gastric cancer, colon cancer, colorectal cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), primary peritoneal cancer, bladder cancer, renal cancer, or or kidney cancer (e.g., renal cell carcinoma), urothelial carcinoma, breast cancer (e.g., Her2-positive breast cancer or triple-negative breast cancer), ovarian cancer, fallopian carcinoma, cervical cancer, uterine cancer, prostate cancer, testicular cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), brain cancer, neuroblastoma, glioblastoma, glioma, astrocytoma, schwannoma, mesothelioma, fibrosarcoma, rhabdomyosarcoma, osteosarcoma, Kaposi's sarcoma, seminoma, and teratocarcinoma. In some embodiments, the combination therapy of the present disclosure is used to treat non-small cell lung cancer, prostate cancer (e.g., prostate adenocarcinoma), glioblastoma, hepatocellular carcinoma, ovarian cancer, head and neck cancer, urothelial carcinoma, or colorectal cancer.

[0117] The combination therapies of the present disclosure are also useful for inhibiting cyclosporine-mediated malignancies or cancer progression (eg, metastasis).

[0118] In some embodiments, the combination therapy of the present disclosure is useful for treating cancer at an early, intermediate, advanced, or metastatic stage. In some embodiments, the combination therapy is used to treat patients (e.g., multiple myeloma patients) who have received at least one previous therapy. In some embodiments, the combination therapy is useful for treating recurrent or refractory cancer.

[0119] In some embodiments, the combination therapy of the present disclosure is used to treat any condition described herein (e.g., a cancer described herein) in a patient who has progressed after standard therapy for said condition or for whom there is no effective standard therapy for said condition.

[0120] In some embodiments, the combination therapy of the present disclosure is used to treat any condition described herein (e.g., a cancer described herein) in a patient who is resistant to one or more standard therapies for said condition. In certain embodiments, the patient is refractory to treatment with Ab2, Ab3, Ab4, galunisertib, LY3200882, XOMA089, daratumumab, MOR202, TAK-079, TAK-573, TAK-169, HexaBody®-CD38, or any combination thereof. The patient may have a condition (e.g., cancer, e.g., multiple myeloma) that is refractory to treatment with an agent that specifically binds to CD38. The condition is refractory to treatment with an agent that specifically binds to CD38. In certain embodiments, the patient may have a condition (e.g., cancer, e.g., multiple myeloma) that is refractory to treatment with Ab2 or daratumumab, or to both treatments.

[0121] In some embodiments, the combination therapy of the present disclosure is used to treat a cancer described herein, wherein the cancer exhibits high levels of TGF-β expression. In certain embodiments, the cancer exhibits high levels of TGF-β expression and is resistant to treatment with an agent that specifically binds to CD38 (e.g., Ab2, Ab3, Ab4, daratumumab, MOR202, TAK-079, TAK-573, TAK-169, HexaBody®-CD38, or any combination thereof). In certain embodiments, the cancer is refractory to treatment with Ab2 or daratumumab, or to both treatments.

[0122] In some embodiments, the combination therapy of the present disclosure is used to treat patients with newly diagnosed multiple myeloma, and the patient is unable to receive a type of stem cell transplant using their own stem cells (autologous stem cell transplant). Additionally or alternatively, the patient has received at least one previous medication for treating multiple myeloma. In certain embodiments, the combination therapy further comprises: a) lenalidomide and / or dexamethasone, b) bortezomib, lenalidomide, and / or dexamethasone; c) bortezomib, melphalan, and / or prednisone, or d) Bortezomib and / or dexamethasone.

[0123] In some embodiments, the combination therapy of the present disclosure is used to treat patients who have received at least two prior medications for treating multiple myeloma. In certain embodiments, the two prior medications include lenalidomide and / or a proteasome inhibitor.

[0124] In some embodiments, the combination therapy of the present disclosure is used to treat patients who have received at least three prior medications for treating multiple myeloma. In certain embodiments, the three prior medications include a proteasome inhibitor and / or an immunomodulatory agent.

[0125] In some embodiments, the combination therapy of the present disclosure is used to treat patients who have failed to respond to proteasome inhibitors and / or immunomodulatory drugs.

[0126] In some embodiments, the combination therapy of the present disclosure is used to treat patients with relapsed / refractory multiple myeloma with a combination of pomalidomide and / or dexamethasone (e.g., low-dose dexamethasone). In certain embodiments, the patient has received at least two prior medications for treating multiple myeloma. In certain embodiments, the two prior medications include lenalidomide and / or a proteasome inhibitor.

[0127] "Treat," "treating," and "treatment" refer to a method of alleviating or abrogating a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, "treatment" as referred to herein includes reference to curative, palliative, and prophylactic treatment. It will be understood that in the context of cancer therapy, "treatment" includes any medical procedure that results in a slowing of cancer growth, delaying cancer progression or recurrence, or reduction in cancer metastasis, as well as partial remission of cancer, in order to extend the patient's life expectancy.

[0128] As used herein, the terms "co-administration," "co-administered," and "in combination" refer, without limitation, to (i) simultaneous administration of therapeutic agents to a patient in need of treatment when such agents are formulated together in a single dosage form; (ii) substantially simultaneous administration of such agents to a patient in need of treatment when such agents are formulated separate from one another in separate dosage forms; and (iii) sequential administration of such agents to a patient in need of treatment when such agents are formulated separate from one another in separate dosage forms that are taken by the patient at different times.

[0129] The ratio between an agent that specifically binds TGF-β (e.g., an anti-TGF-β antibody) and an agent that specifically binds CD38 (e.g., an anti-CD38 antibody) is such that the agents are administered in equal amounts (i.e., a 1:1 ratio), although this is not necessarily the case. Depending on the characteristics of the individual agents, it may be desirable to use unequal amounts of the agents.

[0130] It is understood that the combination therapies of the present disclosure are for use in the methods of treatment described herein, for use in the treatments described herein, and / or for use in the manufacture of medicaments for the treatments described herein.

[0131] Dosing regimen The combination therapy of the present disclosure is administered in an effective amount (i.e., dosage and duration) for treating the condition in question necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex, health, and weight of the patient, and whether the administered agent is administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments.

[0132] A "therapeutically effective amount" refers to the amount of a therapeutic agent administered that will relieve to some extent one or more symptoms of the disorder being treated. This amount is determined by a healthcare professional using well-established principles. A therapeutically effective amount of an anti-cancer therapy may result in, for example, tumor shrinkage, increased viability, elimination of cancer cells, reduced disease progression, reversal of metastasis, or other clinical endpoint desired by a healthcare professional.

[0133] In some embodiments, patients are monitored for cardiac and pulmonary side effects when treated with the combination therapy of the present disclosure.

[0134] Host cells and methods for producing antibodies and antibody compositions One aspect of the present disclosure relates to methods for producing antibodies for the present combination therapy. One embodiment relates to a method for producing the antibodies described herein, comprising providing a recombinant host cell capable of expressing the antibody, culturing the host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody. The antibody produced by such expression in such a recombinant host cell is referred to herein as a "recombinant antibody." Progeny of such host cells and the antibodies produced therefrom are also described.

[0135] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. A host cell can contain, for example, one or more of the vectors described herein. A host cell can contain, for example, a nucleotide sequence encoding the heavy chain or an antigen-binding fragment thereof, a nucleotide sequence encoding the light chain or an antigen-binding fragment thereof, or both, of an anti-TGF-β and / or anti-CD38 antibody or antigen-binding fragment thereof described herein. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of such a cell. Certain modifications may occur later due to mutation or environmental influences. Because such progeny may not, in fact, be identical to the parent cell, because they may have been produced over several generations, they are still included within the term "host cell" as used herein.

[0136] In some embodiments, a host cell of the present disclosure comprises: - a nucleotide sequence encoding the heavy chain or an antigen-binding fragment thereof, a nucleotide sequence encoding the light chain or an antigen-binding fragment thereof, or both, of an anti-TGF-β antibody described herein; and - a nucleotide sequence encoding the heavy chain or an antigen-binding fragment thereof, the nucleotide sequence encoding the light chain or an antigen-binding fragment thereof, or both, of an anti-CD38 antibody described herein.

[0137] Nucleic acid molecules encoding the heavy and / or light chain amino acid sequences of anti-TGF-β and / or anti-CD38 antibodies or antigen-binding fragments thereof are contained in an expression vector. Expression vectors in which the nucleic acid sequence of interest is linked to the necessary expression control sequences (e.g., transcriptional and translational control sequences) include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (e.g., cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, etc.). The antibody light chain coding sequence and the antibody heavy chain coding sequence are inserted into separate vectors and operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, sequences encoding both are inserted into the same expression vector and operably linked to the same expression control sequence (e.g., a common promoter), separate identical expression control sequences (e.g., promoters), or different expression control sequences (e.g., promoters). The antibody coding sequence is inserted into the expression vector by standard methods (ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).

[0138] Expression vectors encoding the antibodies and antigen-binding fragments described herein are introduced into host cells for expression. In some embodiments, the anti-TGF-β antibody-encoding expression vector and the anti-CD38 antibody-encoding expression vector are introduced into separate host cells. In other embodiments, the expression vectors are introduced into the same host cell. The host cells are cultured under conditions appropriate for antibody expression, followed by recovery and isolation. Host cells include mammalian, plant, bacterial, or yeast host cells. Mammalian cell lines available as expression hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma-derived cells (e.g., Hep G2), A549 cells, and several other cell lines. Cell lines are selected based on their expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells.

[0139] Furthermore, antibody expression can be enhanced using several known techniques, for example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions.

[0140] Tissue culture media for host cells may or may not contain animal-derived components (ADCs), such as bovine serum albumin. In some embodiments, ADC-free culture media is preferred for human safety. Tissue culture is performed using fed-batch, continuous perfusion, or any other method appropriate for the host cells and desired yield.

[0141] In some embodiments, the present disclosure relates to a method for producing an antibody composition comprising an anti-TGF-β antibody, or antigen-binding fragment thereof, and an anti-CD38 antibody, or antigen-binding fragment thereof, the method comprising: - providing first and second host cells, wherein the first host cell is capable of expressing an anti-TGF-β antibody or antigen-binding fragment thereof described herein and the second host cell is capable of expressing an anti-CD38 antibody or antigen-binding fragment thereof described herein; - culturing the first and second host cells under conditions suitable for expression of the anti-TGF-β antibody, or antigen-binding fragment thereof, and the anti-CD38 antibody, or antigen-binding fragment thereof; - isolating the resulting antibodies or antigen-binding fragments; and - optionally combining antibodies or antigen-binding fragments to produce an antibody composition.

[0142] Pharmaceutical Composition Another aspect of the present disclosure is a pharmaceutical composition comprising, as active ingredients (e.g., as the only active ingredients), an agent that specifically binds human TGF-β (e.g., an anti-TGF-β antibody or antigen-binding fragment thereof) and an agent that specifically binds human CD38 (e.g., an anti-CD38 antibody or antigen-binding fragment thereof). The agent that specifically binds TGF-β and the agent that specifically binds CD38 are co-formulated (e.g., mixed) and provided in a single composition. The present disclosure also provides (1) a pharmaceutical composition comprising an agent that specifically binds human TGF-β and (2) a pharmaceutical composition comprising an agent that specifically binds human CD38, used in the same combination therapy.

[0143] In some embodiments, when administered in the combination therapy of the present disclosure, the pharmaceutical compositions described herein are intended for the treatment (e.g., amelioration and / or prevention) of disorders, diseases, or conditions by regulating the activity or expression of TGF-β and CD38, thereby improving or slowing the progression of the disorder, disease, or condition. In certain embodiments, the pharmaceutical compositions are intended for the treatment (e.g., amelioration and / or prevention) of cancer. In certain embodiments, the cancer is multiple myeloma.

[0144] In some embodiments, pharmaceutical compositions of the present disclosure comprise an anti-TGF-β antibody described herein that is less than 1% half antibody. Half antibody formation is determined, for example, through purity analysis of a monoclonal antibody preparation by using SDS-capillary electrophoresis under non-reducing conditions or non-reducing SDS-PAGE analysis followed by concentration measurement or RP-HPLC (Angal et al., Mol Immunol 30(1):105-8). (1993);Bloom et al., Protein Science 6:407~415 (1997);Schuurman et al., 38(1):1~8 (2001); and Solanos et al., Anal Chem 78:6583~94 (2006)).

[0145] Generally, the pharmaceutical compositions described herein are suitable for administration as a formulation together with one or more pharmaceutically acceptable excipients, e.g., as described below.

[0146] The terms "excipient" or "carrier" are used herein to describe any ingredient other than the compound of the disclosure. The choice of excipient will largely depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. "Pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, Agents that may be used include, but are not limited to, antibacterial and antifungal agents, isotonicity and absorption delaying agents, etc. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerin, ethanol, etc., and combinations thereof. In some cases, isotonicity agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody. In some embodiments, the pharmaceutical compositions of the present disclosure comprise hyaluronidase (e.g., recombinant human hyaluronidase). In certain embodiments, the pharmaceutical compositions comprising hyaluronidase are for subcutaneous administration.

[0147] The pharmaceutical compositions of the present disclosure are prepared, packaged, or provided in bulk as a single unit dose or as a plurality of single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of each active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject or a convenient fraction of such a dose, for example, half or one-third of such a dose.

[0148] The pharmaceutical compositions of the present disclosure are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physically breaking through the target tissue and penetrating the tissue, thus generally resulting in direct administration into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intratumoral, and intrasynovial injection or infusion; as well as kidney dialysis infusion techniques. Regional perfusion is also intended. Preferred embodiments may include intravenous and subcutaneous routes.

[0149] Pharmaceutical compositions suitable for parenteral administration typically contain an active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations are prepared, packaged, or provided in a form suitable for bolus or continuous administration. Injectable formulations are prepared, packaged, or provided in unit dosage form, for example, in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further contain one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with an appropriate vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations may also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (e.g., having a pH of 3 to 9); however, depending on the application, they are more appropriately formulated as sterile nonaqueous solutions or dried (e.g., lyophilized) forms used with an appropriate vehicle, such as sterile pyrogen-free water. Exemplary parenteral dosage forms include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextrose solutions. Such dosage forms are suitably buffered, if necessary. Other useful parenterally administrable formulations include those containing the active ingredient in microcrystalline form or in a liposomal preparation. Formulations for parenteral administration are formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release.

[0150] Products and Kits The present disclosure also provides agents that specifically bind to human TGF-β and agents that specifically bind to human CD38. In some embodiments, the article of manufacture of the present disclosure comprises an anti-TGF-β antibody or antigen-binding fragment thereof described herein and an anti-CD38 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the article of manufacture comprises Ab1 and Ab2. The present disclosure further provides methods of making the article of manufacture.

[0151] The present disclosure also provides a kit comprising an agent that specifically binds to human TGF-β and an agent that specifically binds to human CD38, as well as instructions for using the combined agent. In some embodiments, the kit of the present disclosure comprises an anti-TGF-β antibody or antigen-binding fragment thereof described herein and an anti-CD38 antibody or antigen-binding fragment thereof described herein. In certain embodiments, the kit comprises Ab1 and Ab2.

[0152] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control.

[0153] Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and common used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as generally accomplished in the art or as described herein.

[0154] Further, unless otherwise required by context, the singular includes the plural and the plural includes the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" will be understood to imply the inclusion of a given integer or group of integers, but not the exclusion of any other integer or group of integers.

[0155] All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. [Example]

[0156] In order that the present disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0157] Example 1: TGF-β is released by multiple myeloma and lymphoma cell lines JJN3, NCI-H929, RPMI8226, LP1, MOLP8, SUDHL-4, DAUDI, OCI-LY19, and SUDHL8 cells were cultured in RPMI1640 (Invitrogen catalog no. 22400-089) supplemented with 10% fetal bovine serum (Invitrogen catalog no. 10082-147).

[0158] To quantify TGF-β released by MM and lymphoma cell lines, 1 × 10 6 Cells of each cell line were plated in one 96-well tissue culture treated plate in a volume of 100 μL of RPMI supplemented with 10% fetal bovine serum. Cells were incubated in a 5% CO atmosphere containing The plates were incubated at 37°C for 1, 2, or 3 days in a humidified incubator. After centrifugation at 250g for 5 minutes, 40 μL of supernatant from each well was collected, and total human TGF-β levels were determined using an MSD 96-well Multi-Array Human TGF-β1 Assay Kit (Meso Scale Discovery catalog number K151IUC-1) according to the manufacturer's instructions. Briefly, samples were activated by acid treatment, followed by neutralization, and then incubation with a capture antibody. The antibody was then detected using an ELISA-based method, and results were read on an MSD SECTOR Imager.

[0159] Total TGF-β was detected at levels above 500 pg / mL in the supernatants of all MM and lymphoma cell lines, and prolonged accumulation was observed in most cell lines, particularly in JJN3, RPMI8226, and MOLP8 cells (Fig. 1).

[0160] Example 2: TGF-β reduces the cytolytic activity of human NK cells and diminishes Ab2-mediated ADCC NK92V cells (NK92-05 CD16 V / V; Conkwest, Inc.) in the logarithmic growth phase were suspended in growth medium (Middle MyeloCult H5100 (STEMCELL Technologies Inc. catalog number 05150) supplemented with 100 units / mL human interleukin-2 (R&D Systems, Inc. catalog number 202-IL-010 / CF)) and plated at 0.8 × 10 cells per mL in a 10-cm dish. 5 Cells were treated with recombinant human TGF-β at a final concentration of 10 ng / mL overnight, 24 hours, 48 ​​hours, or 72 hours at 37°C.

[0161] MOLP8 cells were cultured in RPMI1640 (Invitrogen Catalog No. 22400-089) supplemented with 20% fetal bovine serum (Invitrogen Catalog No. 10082-147).

[0162] Calcein (AM) (Invitrogen Cat. No. C3100MP)-labeled MOLP8 target cells and NK92V effector cells were plated in a 96-well plate at 4 × 10 per well. 4 target cells (MOLP8) and 1.2 × 10 5 Effector cells (NK92V) were seeded at a density of 1000x1000x1000. Cells were treated with Ab2 at concentrations of 0, 0.001, 0.1, or 1 μg / mL, or with an IgG1 control (data not shown), for 1 hour at 37°C. Calcein fluorescence released from lysed target cells in the supernatant was measured using a multimode plate reader (EnVision). The intensity of the fluorescent signal was directly proportional to the number of lysed target cells.

[0163] Incubation of human NK cells with TGF-β for up to 72 hours resulted in a time-dependent decrease in Ab2-mediated ADCC (E:T = 3:1). NK cells incubated with TGF-β for 24 hours retained their lytic activity, whereas lysis by NK cells incubated for 48 or 72 hours was reduced by approximately 25-60% (Figure 2).

[0164] Example 3: TGF-β neutralization restores NK cell lytic activity and Ab2-mediated ADCC NK92V cells (NK92-05 CD16 V / V; Conkwest, Inc.) in the logarithmic growth phase were suspended in growth medium (Middle MyeloCult H5100 (STEMCELL Technologies Inc. catalog number 05150) supplemented with 100 units / mL human interleukin-2 (R&D Systems, Inc. catalog number 202-IL-010 / CF)) and plated at 0.8 × 10 cells per mL in a 10-cm dish. 5 Cells were seeded at a density of 1 / 4 cells / well. Cells were treated with recombinant human TGF-β at a final concentration of 10 ng / mL and either 1D11 (a murine surrogate of Ab1) or an antibody isotype counter. The control (13C4; data not shown) was treated at a final concentration of 50 μg / mL for 90 hours at 37°C.

[0165] Calcein (AM) (Invitrogen Cat. No. C3100MP)-labeled MOLP8 target cells and NK92V effector cells were plated in a 96-well plate at 4 × 10 per well. 4 target cells (MOLP8) and 1.2 × 10 5 Effector cells (NK92V) were seeded at a density of 0.001, 0.01, 0.1, or 1 μg / mL of Ab2 or 1 μg / mL of IgG1 control for 1 hour at 37°C. Calcein fluorescence released from lysed target cells in the supernatant was measured using a multimode plate reader (EnVision). The intensity of the fluorescent signal was directly proportional to the number of lysed target cells.

[0166] The decrease in lytic activity of NK cells and Ab2-mediated ADCC induced by TGF-β was completely restored when TGF-β was neutralized by Ab1, but not by a control antibody (Fig. 3).

[0167] Example 4: TGF-β inhibits Ab2-mediated ADCC of NCI-H929 cells, and Ab1 blocks this inhibition Human NK cells were isolated by negative selection (STEMCELL Technologies Inc.) from normal human donors. The enriched NK cells were cultured in the presence of IL-2 (100 IU / mL), TGF-β (as indicated), and / or Ab1 or isotype control for 3 days at 37°C in a humidified incubator. At this time, exponentially growing NCI-H929 cells were labeled with calcein (AM) and incubated with Ab2 at 1 μg / mL for 30 minutes. The NK effector cells were then resuspended and combined with labeled target NCI-H929 cells for 1 hour in a humidified incubator. Ab2-mediated ADCC was quantified by measuring the level of calcein (AM)-labeled target cells using a spectrophotometer. Increasing doses of TGF-β inhibited the ability of normal human NK cells to kill Ab2-pretreated NCI-H929 cells, with 0.1 ng / mL having little effect on NK cell-mediated cytolysis, whereas 1 and 10 ng / mL blocked ADCC by approximately 50% (Figure 4). ADCC was then assessed at two different concentrations of Ab1 (100 and 200 μg / mL). When added to cultures in the absence of TGF-β, neither the isotype control Ab (IgG4) nor Ab1 had any effect on ADCC (Figures 5 and 6). Ab1 blocked the ability of TGF-β to inhibit ADCC (Figures 5 and 6, p<0.005 vs. untreated), whereas the IgG4 control was unable to block the effects of TGF-β to the same extent. Thus, these data demonstrate that Ab1 can attenuate the immunosuppressive effects of TGF-β on NK cell-mediated ADCC.

[0168] Example 5: Neutralization of endogenous TGF-β by Ab1 restores primary NK cell lytic activity and Ab2-mediated ADCC JJN3, K562, and RPMI8226 cells were cultured in RPMI1640 (Invitrogen Catalog No. 22400-089) supplemented with 10% fetal bovine serum (Invitrogen Catalog No. 10082-147).

[0169] Human primary NK cells were isolated by negative selection from normal human PBMCs according to the manufacturer's suggested protocol (STEMCELL Technologies Inc., catalog number 17955RF). Isolated NK cells were cultured alone or in coculture with JJN3 cells in 6-transwell plates in the presence or absence of 100 mg / ml Ab1 or isotype control for 90 hours at 37°C in a 5% CO2 incubator.

[0170] After 90 hours of co-culture, NK cells from the transwells were incubated for 2 hours at 37°C with calcein (AM) (Invitrogen catalog no. C3100MP) to detect cytolytic function of NK cells, or with 0.001 or 0.1 mg / ml of Ab2, or 0.1 mg / ml of control Ab2 variant (Ab) in calcein (AM)-labeled RPMI 8226 to detect Ab2-mediated ADCC. * The calcein was incubated with labeled K562 cells in the presence of 1000 calcein-containing IgG4 (NaCl) for 1 hour at 37°C. The calcein fluorescence released from lysed target cells in the supernatant was measured using a multimode plate reader (EnVision). The intensity of the fluorescent signal was directly proportional to the number of lysed target cells.

[0171] The increased cytolytic activity of primary NK cells (Fig. 7) and increased Ab2-mediated ADCC (Fig. 8) demonstrate that the presence of Ab1 neutralized JJN3 cell-released endogenous TGF-β during the 90-hour coculture.

[0172] Example 6: Combination Treatment with Ab1 and Ab2 in Patients with Multiple Myeloma The effect of combination treatment with antibodies Ab1 and Ab2 was further evaluated in human patients, e.g., multiple myeloma patients, who were resistant to one or more other treatments that target CD38 (e.g., isatuximab or daratumumab), e.g., who were unresponsive to or had progressed during treatment with other treatments that target CD38.

[0173] The combination of Ab1 and Ab2 is expected to treat multiple myeloma more effectively than Ab2 alone, e.g., combination treatment results in improved symptoms, reduced bone destruction, enhanced bone formation, bone remodeling and / or fracture healing, delayed cancer progression or recurrence, or increased life expectancy compared to treatment with Ab2 alone.

[0174] Ab1 and Ab2 were administered using multiple doses (e.g., 1-20 mg / kg), dosing schedules (e.g., every 1, 2, 3, or 4 weeks), for the period necessary to achieve the desired results, as determined, for example, by a physician. In some embodiments, one or both antibodies were administered via intravenous infusion. In particular embodiments, Ab2 was administered at 10 mg / kg or 16 mg / kg actual body weight every 1, 2, 3, or 4 weeks, or any combination thereof, over the course of treatment.

[0175] List of sequences SEQ ID NO: 1 (Ab1 heavy chain) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK SEQ ID NO: 2 (Ab1 light chain) ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC SEQ ID NO: 3 (Ab1 heavy chain variable domain) QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SNVISWVRQA PGQGLEWMGG VIPIVDIANY AQRFKGRVTI TADESTSTTY MELSSLRSED TAVYYCASTL GLVLDAMDYW GQGTLVTVSS SEQ ID NO: 4 (Ab1 light chain variable domain) ETVLTQSPGT LSLSPGERAT LSCRASQSLG SSYLAWYQQK PGQAPRLLIY GASSRAPGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYADSPITFG QGTRLEIK SEQ ID NO: 5 (Ab1 heavy chain CDR1) SNVIS SEQ ID NO: 6 (Ab1 heavy chain CDR2) GVIPIVDIAN Y SEQ ID NO: 7 (Ab1 heavy chain CDR3) TLGLVLDAMD Y SEQ ID NO: 8 (Ab1 light chain CDR1) RASQSLGSSY LA SEQ ID NO: 9 (Ab1 light chain CDR2) GASSRAP SEQ ID NO: 10 (Ab1 light chain CDR3) QQYADSPIT SEQ ID NO: 11 (Ab2 heavy chain) QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 12 (Ab2 light chain) DIVMTQSHLS MSTSLGDPVS ITCKASQDVS TVVAWYQQKP GQSPRRLIYS ASYRYIGVPD RFTGSGAGTD FTFTISSVQA EDLAVYYCQQ HYSPPYTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO: 13 (Ab2 heavy chain variable domain) QVQLVQSGAE VAKPGTSVKL SCKASGYTFT DYWMQWVKQR PGQGLEWIGT IYPGDGDTGY AQKFQGKATL TADKSSKTVY MHLSSLASED SAVYYCARGD YYGSNSLDYW GQGTSVTVSS SEQ ID NO: 14 (Ab2 light chain variable domain) DIVMTQSHLS MSTSLGDPVS ITCKASQDVS TVVAWYQQKP GQSPRRLIYS ASYRYIGVPD RFTGSGAGTD FTFTISSVQA EDLAVYYCQQ HYSPPYTFGG GTKLEIK SEQ ID NO: 15 (Ab2 heavy chain CDR1) DYWMQ SEQ ID NO: 16 (Ab2 heavy chain CDR2) TIYPGDGDTG YAQKFQG SEQ ID NO: 17 (Ab2 heavy chain CDR3) GDYYGSNSLD Y SEQ ID NO: 18 (Ab2 light chain CDR1) KASQDVSTVV A SEQ ID NO: 19 (Ab2 light chain CDR2) SASYRYI SEQ ID NO: 20 (Ab2 light chain CDR3) QQHYSPPYT SEQ ID NO: 21 (human TGF-β1): SwissProt P01137 MPPSGLRLLL LLLPLLWLLV LTPGRPAAGL STCKTIDMEL VKRKRIEAIR GQILSKLRLA SPPSQGEVPP GPLPEAVLAL YNSTRDRVAG ESAEPEPEPE ADYYAKEVTR VLMVETHNEI YDKFKQSTHS IYMFFNTSEL REAVPEPVLL SRAELRLLRL KLKVEQHVEL YQKYSNNSWR YLSNRLLAPS DSPEWLSFDV TGVVRQWLSR GGEIEGFRLS AHCCSCDSRDN TLQVDINGFT TGRRGDLATI HGMNRPFLLL MATPLERAQH LQSSRHRRAL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS SEQ ID NO: 22 (human TGF-β2): SwissProt P08112 MHYCVLSAFL ILHLVTVALS LSTCSTLDMD QFMRKRIEAI RGQILSKLKL TSPPEDYPEP EEVPPEVISI YNSTRDLLQE KASRRAAACE RERSDEEYYA KEVYKIDMPP FFPSENAIPP TFYRPYFRIV RFDVSAMEKN ASNLVKAEFR VFRLQNPKAR VPEQRIELYQ ILKSKDLTSP TQRYIDSKVV KTRAEGEWLS FDVTDAVHEW LHHKDRNLGF KISLHCPCCT FVPSNNYIIP NKSEELEARF AGIDGTSTYT SGDQKTIKST RKKNSGKTPH LLLMLLPSYR LESQQTNRRK KRALDAAYCF RNVQDNCCLR PLYIDFKRDL GWKWIHEPKG YNANFCAGAC PYLWSSDTQH SRVLSLYNTI NPEASASPCC VSQDLEPLTI LYYIGKTPKI EQLSNMIVKS CKCS SEQ ID NO: 23 (human TGF-β3): SwissProt P10600 MKMHLQRALV VLALLNFATV SLSLSTCTTL DFGHIKKKRV EAIRGQILSK LRLTSPPEPT VMTHVPYQVL ALYNSTRELL EEMHGEREEG CTQENTESEY YAKEIHKFDM IQGLAEHNEL AVCPKGITSK VFRFNVSSVE KNRTNLFRAE FRVLRVPNPS SKRNEQRIEL FQILRPDEHI AKQRYIGGKN LPTRGTAEWL SFDVTDTVRE WLLRRESNLG LEISIHCPCH TFQPNGDILE NIHEVMEIKF KGVDNEDDHG RGDLGRLKKQ KDHHNPHLIL MMIPPHRLDN PGQGGQRKKR ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS SEQ ID NO: 24 (human CD38): GenBank NP_001766 MANCEFSPVS GDKPCCRLSR RAQLCLGVSI LVLILVVVLA VVVPRWRQQW SGPGTTKRFP ETVLARCVKY TEIHPEMRHV DCQSVWDAFK GAFISKHPCN ITEEDYQPLM KLGTQTVPCN KILLWSRIKD LAHQFTQVQR DMFTLEDTLL GYLADDLTWC GEFNTSKINY QSCPDWRKDC SNNPVSVFWK TVSRRFAEAA CDVVHVMLNG SRSKIFDKNS TFGSVEVHNL QPEKVQTLEA WVIHGGREDS RDLCQDPTIK ELESIISKRN IQFSCKNIYR PDKFLQCVKN PEDSSCTSEI SEQ ID NO: 25 (Ab3 heavy chain) QVQLVQSGAE VVKPGASVKV SCKASGYTFT SYAMHWVKEA PGQRLEWIGY IYPGQGGTNY NQKFQGRATL TADTSASTAY MELSSLRSED TAVYFCARTG GLRRAYFTYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLAG PDVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPLPEEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG SEQ ID NO: 26 (Ab3 light chain) DIVLTQSPAT LSLSPGERAT ISCRASQSVS SYGQGFMHWY QQKPGQPPRL LIYGASSRAT GIPARFSGSG SGTDFTLTIS PLEPEDFAVY YCQQNKEDPW TFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID NO: 27 (Ab3 heavy chain variable domain) QVQLVQSGAE VVKPGASVKV SCKASGYTFT SYAMHWVKEA PGQRLEWIGY IYPGQGGTNY NQKFQGRATL TADTSASTAY MELSSLRSED TAVYFCARTG GLRRAYFTYW GQGTLVTVSS SEQ ID NO: 28 (Ab3 light chain variable domain) DIVLTQSPAT LSLSPGERAT ISCRASQSVS SYGQGFMHWY QQKPGQPPRL LIYGASSRAT GIPARFSGSG SGTDFTLTIS PLEPEDFAVY YCQQNKEDPW TFGGGTKLEI K SEQ ID NO: 29 (Ab3 heavy chain CDR1) GYTFTSYA SEQ ID NO: 30 (Ab3 heavy chain CDR2) IYPGQGGT SEQ ID NO: 31 (Ab3 heavy chain CDR3) ARTGGLRRAY FTY SEQ ID NO: 32 (Ab3 light chain CDR1) QSVSSYGQGF SEQ ID NO: 33 (Ab3 light chain CDR2) GAS SEQ ID NO: 34 (Ab3 light chain CDR3) QQNKEDPWT SEQ ID NO: 35 (Ab4 heavy chain) QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY ADSVKGRFTI SGDNSKNTLY LQMNSLRAED TAVYYCARMF RGAFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APELLAGPDV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPL PEEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG SEQ ID NO: 36 (Ab4 light chain) AIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISGLQP EDSATYYCLQ DYIYYPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO: 37 (Ab4 heavy chain variable domain) QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAV IWYDGSNKYY ADSVKGRFTI SGDNSKNTLY LQMNSLRAED TAVYYCARMF RGAFDYWGQG TLVTVSS SEQ ID NO: 38 (Ab4 light chain variable domain) AIQMTQSPSS LSASVGDRVT ITCRASQGIR NDLGWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISGLQP EDSATYYCLQ DYIYYPTFGQ GTKVEIK SEQ ID NO: 39 (Ab4 heavy chain CDR1) GFTFSSYG SEQ ID NO: 40 (Ab4 heavy chain CDR2) IWYDGSNK SEQ ID NO: 41 (Ab4 heavy chain CDR3) ARMFRGAFDY SEQ ID NO: 42 (Ab4 light chain CDR1) QGIRND SEQ ID NO: 43 (Ab4 light chain CDR2) AAS SEQ ID NO: 44 (Ab4 light chain CDR3) LQDYIYYPT

Claims

1. 1. A method of treating cancer in a human patient in need thereof, comprising administering to the patient an anti-CD38 antibody having a heavy chain CDR1 (HCDR1), HCDR2, HCDR3, a light chain CDR1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 15-20, respectively, and an anti-TGF-β antibody or antigen-binding fragment thereof having a HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 5-10, respectively.

2. The anti-CD38 antibodies each comprise a heavy chain variable domain (V Ｈ ) and a light chain variable domain (V Ｌ ) comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; Ｈ and V Ｌ The method of claim 1 , comprising:

3. The method of claim 2, wherein the anti-CD38 antibody has a heavy chain (HC) and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively; and the anti-TGF-β antibody has a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

4. A method of treating cancer in a human patient in need thereof, comprising administering to the patient an anti-CD38 antibody and an anti-TGF-β antibody, or antigen-binding fragment thereof.

5. The anti-CD38 antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs: 15-20, respectively; b) a heavy chain variable domain (V) comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively; Ｈ ) and a light chain variable domain (V Ｌ ) or c) having a heavy chain (HC) and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively.

6. The anti-TGF-β antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs:5-10, respectively; b) V comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively Ｈ and V Ｌ or c) having a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

7. Anti-CD38 antibody is human IgG １ The anti-TGF-β antibody contains a human IgG Fc region. ４ The method of any one of claims 1, 2, or 4 to 6, comprising an Fc region.

8. A method for treating multiple myeloma in a human patient in need thereof, comprising administering to a patient a heavy chain variable domain (V Ｈ ) amino acid sequence and the light chain variable domain of SEQ ID NO: 14 (V Ｌ ) an anti-CD38 antibody comprising the amino acid sequence of SEQ ID NO:3; Ｈ Amino acid sequence and V of SEQ ID NO:4 Ｌ The method comprises administering to the patient an anti-TGF-β antibody comprising the amino acid sequence.

9. A method of treating multiple myeloma in a human patient in need thereof, comprising administering to the patient an anti-CD38 antibody comprising a heavy chain (HC) amino acid sequence of SEQ ID NO:11 and a light chain (LC) amino acid sequence of SEQ ID NO:12, and an anti-TGF-β antibody comprising a HC amino acid sequence of SEQ ID NO:1 and a LC amino acid sequence of SEQ ID NO:

2.

10. The method of any one of claims 1 to 9, wherein the anti-CD38 antibody and the anti-TGF-β antibody or fragment are administered sequentially to the patient.

11. An anti-CD38 antibody in combination with an anti-TGF-β antibody for use in treating cancer in a human patient in need thereof.

12. Use of an anti-CD38 antibody in combination with an anti-TGF-β antibody for the manufacture of a medicament for treating cancer in a human patient in need thereof.

13. The anti-CD38 antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs: 15-20, respectively; b) V comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively Ｈ and V Ｌ or c) An antibody for use according to claim 11 or the use according to claim 12, having a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively.

14. An anti-TGF-β antibody in combination with an anti-CD38 antibody for use in treating cancer in a human patient in need thereof.

15. Use of an anti-TGF-β antibody in combination with an anti-CD38 antibody for the manufacture of a medicament for treating cancer in a human patient in need thereof.

16. The anti-TGF-β antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs:5-10, respectively; b) V comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively Ｈ and V Ｌ or c) An antibody for use according to claim 14 or the use according to claim 15, having a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

17. The method according to any one of claims 1 to 10, the antibody for use according to any one of claims 11, 13, 14 and 16, or the use according to any one of claims 12, 13, 15 and 16, wherein the cancer is CD38 positive.

18. 17. The method according to any one of claims 1 to 10, the antibody for use according to any one of claims 11, 13, 14 and 16, or the use according to any one of claims 12, 13, 15 and 16, wherein the cancer is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, diffuse large B-cell lymphoma, peripheral T-cell lymphoma, hairy cell leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, melanoma, glioblastoma, lung cancer, squamous cell carcinoma of the skin, colorectal cancer, breast cancer, ovarian cancer, head and neck cancer, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma.

19. The method according to any one of claims 1 to 10, the antibody for use according to any one of claims 11, 13, 14 and 16, or the use according to any one of claims 12, 13, 15 and 16, wherein the cancer is a hematological malignancy.

20. 20. The method, antibody for use, or use of claim 19, wherein the cancer is multiple myeloma.

21. 21. The method, antibody for use, or use of claim 20, wherein the treatment results in less bone destruction than treatment with anti-CD38 antibody alone.

22. 21. The method, antibody for use, or use of claim 20, wherein treatment enhances fracture healing.

23. 21. The method, antibody for use, or use of claim 20, wherein the treatment further comprises dexamethasone.

24. The method of any one of claims 1 to 10, the antibody for use of any one of claims 11, 13, 14 and 16, or the use of any one of claims 12, 13, 15 and 16, wherein the cancer is refractory to treatment with Ab2 or daratumumab, or to treatment with both Ab2 and daratumumab.

25. A product comprising an anti-CD38 antibody and an anti-TGF-β antibody, said product being suitable for treating cancer with the method of any one of claims 1 to 10 in a patient in need thereof.

26. The anti-CD38 antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs: 15-20, respectively; b) V comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively Ｈ and V Ｌ or c) having a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively; The anti-TGF-β antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs:5-10, respectively; b) V comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively Ｈ and V Ｌ or 26. The product of claim 25, having a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

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