Combination therapy targeting CD38 and TGF-beta for cancer

Abstract

The present disclosure relates to combination cancer therapies that target CD38 and TGF-β using antibodies specific for these targets. Compositions useful for the therapies are also provided.

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 a variety of 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. They have similar in vitro activities, but individual knockouts of specific cell types suggest that their roles in vivo 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 (Ibid.). In addition to this canonical signaling cascade, non-classical 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 end result of TGF-β signaling is a crosstalk of all these signaling pathways, which integrates the state and environment of the cell (Ibid.).

[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 is involved in the regulation of NAD + It catalyzes the conversion of 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 mostly in the lymphoid lineage and at different stages of T and B cell development with varying expression levels.

[0007] CD38 is upregulated in 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), hairy cell leukemia (HCL), 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. CD38 expression in hematopoietic malignancies and its correlation with disease progression make 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 blunt 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 of 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 of 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), where 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 is 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 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) 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.

[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) having 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 (VVD) 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; H and V LIn 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; 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 a heavy chain variable domain amino acid sequence of SEQ ID NO: 13 and a light chain variable domain amino acid sequence of SEQ ID NO: 14, and an anti-TGF-β antibody comprising a heavy chain variable domain amino acid sequence of SEQ ID NO: 3 and a 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 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.

[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 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) having 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.

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

[0022] In some embodiments of the methods, antibodies for use, and uses of the 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, squamous cell carcinoma of the skin, 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 antibody uses described herein, the cancer is multiple myeloma.In certain embodiments, the treatment described herein causes less bone destruction and / or enhances bone formation, resulting in bone remodeling and / or fracture healing in multiple myeloma patients than treatment with anti-CD38 antibody alone.

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

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

[0027] In some embodiments of the methods, antibodies for use, and uses of the antibodies described herein, the cancer is refractory to treatment with Ab2 or daratumumab, or to both treatments. 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 is an article of manufacture comprising an anti-CD38 antibody and an anti-TGF-β antibody, said article of manufacture being 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) having 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 description 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. [Diagram 2]FIG. 13 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. [Diagram 3] FIG. 13 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. 13 is a graph showing the effect of 0.1, 1, and 10 ng / mL TGF-β on Ab2-mediated ADCC of NCI-H929 cells. [Diagram 5] Figure 1 shows a graph depicting 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 depicting 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. 13 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 for 90 hours with 100 μg / mL Ab1 or isotype control. [Figure 8]FIG. 13 shows a graph depicting 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 for 90 hours with 100 μg / mL Ab1 or isotype control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 indicated, "TGF-β" refers to human TGF-β herein. Polypeptide sequences for 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 indicated, "CD38" refers to human CD38 herein. 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 the 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 ) is composed of 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 terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments for each region may be according to the IMGT® definition (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or Kabat, Sequences of Proteins of 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] The term "epitope" as used herein 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, e.g., amino acids or carbohydrate or sugar side chains, and generally have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes are "linear" or "conformational". In a linear epitope, all of the 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. It is possible to determine a desired epitope on an antigen and generate antibodies against that epitope using techniques well known in the art. For example, antibodies against a linear epitope can be generated, for example, by immunizing an animal with a peptide that has 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] It can be determined whether an antibody binds to the same epitope as an antibody described herein or competes for binding by using methods known in the art, including but not limited to competitive assays, epitope binning, and alanine scanning. In some embodiments, the test antibody and the 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 can be bound 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 than 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 methods may be used in two directions to assess whether an antibody cross-competes with another antibody (i.e., to determine if a known antibody blocks the 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 from immunization of a mouse with an antigen of interest, or a chimeric antibody based on such a murine antibody), it is possible to replace certain amino acids, especially in the framework and constant regions of the heavy and light chains, in order to avoid or minimize immune responses in humans. Although it is not possible to predict precisely the immunogenicity and thus 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 for therapeutic antibodies is toward humanized or fully human antibodies. Thus, 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 modification of framework regions of variable domain sequences. Although amino acid residues that are part of complementarity determining regions (CDRs) are often not altered in the context of humanization, in certain cases it is 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 other than 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 occurs in a CDR sequence, particularly Asn-Gly, 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 CDR grafting, which involves, for example, for mouse-derived chimeric antibodies, the identification of human germline gene counterparts for mouse variable domain genes and grafting mouse CDR sequences into this framework. The specificity of the antibody's interaction with the target antigen resides primarily in the amino acid residues located in the six CDRs of the heavy and light chains. Thus, the amino acid sequences within the CDRs are much more variable between individual antibodies than the sequences outside the CDRs. Since the CDR sequences are responsible for most of the 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 the CDR sequences from a particular antibody grafted into framework sequences from different antibodies. 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 definition, although more recent publications (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210-225 (2007)) suggest that the IMGT® definition (the international ImMunoGeneTics information system®, www.imgt.org) may improve the results of humanization (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 derived. Back mutations (sometimes also called "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 residues that are buried and have a low degree of 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 altered to human residues.

[0041] In certain cases, it is desirable to modify one or more CDR amino acid residues to improve the binding affinity to the target epitope. This is known as "affinity maturation". Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks 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 includes antibodies that have sequences derived from human genes, but whose sequences have been modified to, for example, reduce immunogenicity, increase affinity, and increase stability. The term includes antibodies that are recombinantly produced in non-human cells, which may provide glycosylation that is not typical of human cells. The term also includes antibodies that are 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 a F(ab')2 fragment, which is a bivalent fragment comprising 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 the C domain. H2 or C H3 In some cases, the antibody fragment may include 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 different species, or (4) does not occur naturally. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the original cell is "isolated" from its naturally associated components. A protein is also substantially free of 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 are determined by any method known in the art. Generally, the class and subclass of the antibody are determined by using an antibody that is specific for a particular class and subclass of the antibody. Such antibodies are commercially available. The class and subclass are determined by ELISA and Western blot and other techniques. Alternatively, the class and subclass are 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 therapy 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 lower 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 WO2018 / 134681, PCT Patent Publication WO2006 / 086469, PCT Patent Publication 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, 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 in, for example, framework regions) compared to Ab1 without losing the 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 NO: 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 lower case (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 that comprises: a) heavy chain CDR1-3 (HCDR1-3) comprising the amino acid sequences of SEQ ID NOs: 5 to 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 that comprises: 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 a combination of any 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 comprising the amino acid sequences of SEQ ID NOs: 5 to 10, respectively; b) 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) A HC comprising the amino acid sequence of SEQ ID NO:1 and a 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 may also be modified, for example, at Kabat residue L248 (e.g., by introducing the mutation L248E) to reduce any undesirable 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 administered in combination 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., 9×10 -9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, 2×10 -9 M, or 1×10 -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) inhibiting 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 the 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 the 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, e.g., 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) restores the cytolytic activity of NK-92 cells following 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 antibody or antigen-binding fragment described herein has 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] Agents that block CD38 In some embodiments, the agent that specifically binds to CD38 used in the combination therapy described herein is an anti-CD38 antibody or an 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 entirety of which is incorporated herein by reference. In some embodiments, the anti-CD38 antibody is produced by the hybridoma cell line deposited at the American Type Culture Collection under the accession number PTA-7670. 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 Co., Ltd.), TAK-169 (Takeda Pharmaceutical Co., Ltd.), HexaBody®-CD38 (Genmab / Janssen), anti-CD38 SIFbody (Momenta Pharmaceuticals, Inc.), or TSK011010 (CASI Pharmaceuticals Inc.).

[0062] In certain embodiments, the anti-CD38 antibody is the 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, without losing the 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 that comprises: 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 a combination of any of the above heavy chains with any of the above light chains.

[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) 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) A HC comprising the amino acid sequence of SEQ ID NO:11 and a LC comprising the amino acid sequence of SEQ ID NO:12.

[0070] In certain embodiments, the anti-CD38 antibody is the 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, without losing the 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 that comprises: 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 a combination of any of the above heavy chains with any of the above light chains.

[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; 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: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 the 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, without losing the 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) 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: 37. 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 that comprises: 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 a combination of any of the above heavy chains with any of the above light chains.

[0085] 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: 39 to 44; 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; 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:38. L ; c) V comprising the amino acid sequence of SEQ ID NO: 37H 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 at 1×10 -8 M or less, e.g., 9 x 10 -9 M, 8×10 -9 M, 7×10 -9 M, 6×10 -9 M, 5×10 -9 M, 4×10 -9 M, 3×10 -9 M, 2×10 -9 M, or 1×10 -9 K of M D In certain embodiments, the anti-CD38 antibody or antigen-binding fragment binds to CD38 at 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, leukemia cells, or multiple myeloma cells. In even more preferred embodiments, 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, such as maytansinoids, small drugs, tomaymycin derivatives, leptomycin derivatives, prodrugs, taxoids, CC-1065 and CC-1065 analogs, or any of the cytotoxic agents described in U.S. Patent 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 L or C H The nucleic acid sequence encoding the V L or V H The nucleic acid molecule encoding is then operably linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class of immunoglobulin molecule. This is accomplished using a vector or nucleic acid molecule containing a CL or CH chain, as described above. For example, an antibody that was originally IgM is class switched to IgG. In addition, class switching is used to convert one IgG subclass to another (e.g., IgG1 to IgG2). The κ light chain constant region is changed to a λ light chain constant region, and so on. A preferred method of producing the antibodies described herein with a desired Ig isotype includes the steps of isolating a nucleic acid molecule encoding the heavy chain of the antibody and a nucleic acid molecule encoding the light chain of the antibody, obtaining a variable domain of the heavy chain, ligating the variable domain of the heavy chain with a constant region of a heavy chain of the desired isotype, expressing the light chain and the ligated heavy chain in a cell, and harvesting an antibody with the desired isotype.

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

[0101] In one embodiment, the antibody may comprise 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, for example, in cases where ligand / receptor interaction is undesirable or in the case of antibody drug conjugates.

[0102] In some embodiments, the antibodies described herein, such as anti-TGF-β and anti-CD38 antibodies, do not have a C-terminal lysine in the heavy chain. The C-terminal lysine is removed during production 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 a combination therapy 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 therapy 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 therapy described herein may take the form of a method of treatment using the agents or pharmaceutical compositions that include the agents.

[0104] The present disclosure also contemplates combination therapies comprising an agent that specifically binds human CD38 (e.g., an anti-CD38 antibody or antigen-binding fragment thereof, e.g., those described herein) and an agent that specifically binds LAP (e.g., 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 or an antigen-binding fragment thereof; 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: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 an anti-CD38 antibody; - V having the amino acid sequences of SEQ ID NOs: 3 and 4, respectively H And V L an anti-TGF-β antibody or antigen-binding fragment thereof comprising: H And V L an anti-CD38 antibody comprising: - 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 disclosure more effective than treatment with an anti-CD38 antibody alone.

[0109] In some embodiments, the combination therapy comprising an agent that specifically binds human TGF-β and an agent that specifically binds human CD38 (e.g., anti-TGF-β antibody and 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, pan-histone deacetylase inhibitor (e.g., panobinostat), retinoic acid (e.g., all-trans retinoic acid), radioimmunotherapy, chemotherapy, etc. 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 the condition targeted by the combination therapy of the present disclosure. For example, when 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 comprising an anti-TGF-β antibody and an anti-CD38 antibody may involve administration of a single composition comprising 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 disease, inflammatory disease, autoimmune disease, or fibrotic condition.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 the determination are obtained through tumor biopsy or through collection of circulating tumor cells. Without wishing to be bound by theory, it is contemplated that agents that specifically bind to CD38 bind to CD38 positive cells and mediate ADCC / CDC in the cells, and agents that specifically bind to TGF-β reduce 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 (possibly not eligible 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 produces less bone destruction in myeloma bone lesions than a 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 produce 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 producing 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., relapsed or refractory primary amyloidosis or light chain amyloidosis), myelodysplastic syndrome (MDS), monoclonal gammopathy, isolated 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 myelogenous 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, and the like. 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 cancer, 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 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 beyond the 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., cancer described herein) in a patient, and the patient is resistant to one or more standard therapies for said condition. In certain embodiments, the patient may have a condition (e.g., cancer, e.g., multiple myeloma) that 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 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 refractory to both treatments.

[0121] In some embodiments, the combination therapy of the present disclosure is used to treat the cancers described herein, wherein the cancer shows high levels of TGF-β expression. In certain embodiments, the cancer shows 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 his / her own stem cells (autologous stem cell transplant). Additionally or alternatively, the patient has received at least one previous medication to treat 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 medicaments to treat multiple myeloma. In certain embodiments, the two prior medicaments 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 medicaments to treat multiple myeloma. In certain embodiments, the three prior medicaments 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 medicaments for treating multiple myeloma. In certain embodiments, the two prior medicaments include lenalidomide and / or a proteasome inhibitor.

[0127] "Treat", "treating" and "treatment" refer to a method of alleviating or arresting 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 slowing the growth of cancer, delaying cancer progression or recurrence, or reducing cancer metastasis, as well as partial remission of cancer, in order to extend the patient's life expectancy.

[0128] The terms "co-administration," "co-administered," and "in combination" as used herein refer, without limitation, to (i) simultaneous administration of therapeutic agents to a patient in need of treatment, where such agents are formulated together in a single dosage form; (ii) substantially simultaneous administration of such agents to a patient in need of treatment, where 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, where such agents are formulated separate from one another in separate dosage forms that are ingested 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 non-equal 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 agent being administered is administered as a stand-alone treatment or in combination with one or more additional anti-cancer treatments.

[0132] "Therapeutically effective amount" refers to the amount of therapeutic agent administered that relieves to some extent one or more symptoms of the disorder being treated. This amount is determined by a health care professional using well-established principles. A therapeutically effective amount of anti-cancer therapy may, for example, cause tumor shrinkage, increased viability, elimination of cancer cells, reduced disease progression, reversal of metastasis, or other clinical endpoints desired by a health care 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 a method 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 said host cell under suitable conditions for the 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." The progeny of such a host cell and the antibody produced therefrom are also described.

[0135] The term "recombinant host cell" (or simply "host cell") as used herein refers to a cell into which a recombinant expression vector has been introduced. A host cell may, for example, contain one or more vectors as described herein. A host cell may, for example, contain a nucleotide sequence encoding a heavy chain or an antigen-binding fragment thereof, a nucleotide sequence encoding a 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 as 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. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

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

[0137] The nucleic acid molecule encoding the heavy and / or light chain amino acid sequence of the anti-TGF-β and / or anti-CD38 antibody or antigen-binding fragment thereof is 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., transcription and translation 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), to separate identical expression control sequences (e.g., promoters), or to 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] The expression vectors encoding the antibodies and antigen-binding fragments described herein are introduced into a host cell 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 the expression of the antibodies, and then harvested and isolated. The host cells include mammalian, plant, bacterial, or yeast host cells. Mammalian cell lines available as hosts for expression 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 cells (COS), 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 may be used are insect cell lines, such as Sf9 or Sf21 cells.

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

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

[0141] In some embodiments, the present disclosure relates to a method of 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 a first and a second host cell, the first host cell capable of expressing an anti-TGF-β antibody, or antigen-binding fragment thereof, described herein, and the second host cell 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 Compositions 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 to human TGF-β (e.g., an anti-TGF-β antibody or antigen-binding fragment thereof) and an agent that specifically binds to human CD38 (e.g., an anti-CD38 antibody or antigen-binding fragment thereof). The agent that specifically binds to TGF-β and the agent that specifically binds to CD38 are co-formulated (e.g., mixed) and provided in a single composition. The present disclosure also provides a pharmaceutical composition comprising (1) an agent that specifically binds to human TGF-β, and (2) an agent that specifically binds to human CD38, used in the same combination therapy.

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

[0144] In some embodiments, the pharmaceutical compositions of the present disclosure comprise anti-TGF-β antibodies described herein that are less than 1% half-antibody. Half-antibody formation is determined through purity analysis of monoclonal antibody preparations, for example, 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 pharma- ceutically acceptable excipients, e.g., as described below.

[0146] The term "excipient" or "carrier" is used herein to describe any ingredient other than the compound of the present disclosure. The choice of excipient largely depends 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, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerin, ethanol, and the like, and combinations thereof. In some cases, isotonic 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 disclosure comprise hyaluronidase (e.g., recombinant human hyaluronidase). In certain embodiments, the pharmaceutical compositions comprising hyaluronidase are for subcutaneous administration.

[0147] The pharmaceutical composition of the present disclosure is 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 active ingredient that would be administered to a subject, or a convenient fraction of such a dose, for example, half or a 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 tissue of a subject and administration of the pharmaceutical composition through tissue penetration, thus generally resulting in administration directly into the bloodstream, muscle, or internal organs. Thus, parenteral administration includes, but is not limited to, administration of the 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. Local perfusion is also intended. Preferred embodiments may include intravenous and subcutaneous routes.

[0149] The formulation of pharmaceutical compositions suitable for parenteral administration typically comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations are prepared, packaged, or provided in a form suitable for bolus administration or continuous administration. Injectable formulations are prepared, packaged, or provided in unit dosage form, such as in ampoules, or in 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 comprise one or more additional components, including, but are not limited to, suspending agents, stabilizing agents, 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 a suitable 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), although for some applications they are more appropriately formulated as sterile non-aqueous solutions or dry (e.g., lyophilized) forms used with a suitable vehicle, such as sterile pyrogen-free water. Exemplary parenteral administration 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 parenterally administrable formulations that are useful include those that contain 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 an article of manufacture comprising an agent that specifically binds human TGF-β and an agent that specifically binds 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 a method of manufacturing 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, and instructions for using the combined agents.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 shall have the meanings that are commonly understood by those skilled in the art.Exemplary methods and materials are described below, but similar or equivalent methods and materials 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, shall 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 well known and common 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. EXAMPLES

[0156] In order to better understand the present disclosure, 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 6Each cell line was 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 at 37°C for 1, 2, or 3 days in a humidified incubator containing 5% CO2. After centrifugation at 250g for 5 minutes, 40 μL of supernatant from each well was harvested and levels of total human TGF-β were determined using the MSD 96-well Multi-Array Human TGF-β1 Assay Kit (Meso Scale Discovery Catalog No. K151IUC-1) according to the manufacturer's instructions. Briefly, samples were activated by acid treatment followed by neutralization and subsequent incubation with capture antibody. Antibody was then detected using an ELISA-based method and results were read on an MSD SECTOR Imager.

[0159] Total TGF-β was detected in the supernatants of all MM and lymphoma cell lines at levels above 500 pg / mL, and long-term 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 decreases Ab2-mediated ADCC NK92V cells (NK92-05 CD16 V / V; Conkwest, Inc.) in the log phase of proliferation were suspended in growth medium (Middle MyeloCult H5100 (STEMCELL Technologies Inc. catalog no. 05150) supplemented with 100 units / mL human interleukin-2 (R&D Systems, Inc. catalog no. 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 for overnight, 24 h, 48 h, or 72 h 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 96-well plates at 4 × 10 per well. 4 Target cells (MOLP8) and 1.2 × 10 5 Effector cells (NK92V) were seeded at a density of 100x1000 cells / mL. Cells were treated with Ab2 at concentrations of 0, 0.001, 0.1, or 1 μg / mL, or IgG1 control (data not shown) for 1 h at 37°C. Released calcein fluorescence 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 h resulted in a time-dependent decrease in Ab2-mediated ADCC (E:T = 3:1). NK cells incubated with TGF-β for 24 h retained their lytic activity, whereas lysis by NK cells incubated for 48 or 72 h 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 log phase of proliferation were suspended in growth medium (Middle MyeloCult H5100 (STEMCELL Technologies Inc. catalog no. 05150) supplemented with 100 units / mL human interleukin-2 (R&D Systems, Inc. catalog no. 202-IL-010 / CF)) and plated at 0.8 × 10 cells per mL in a 10 cm dish. 5Cells were treated with recombinant human TGF-β at a final concentration of 10 ng / mL and either 1D11 (a mouse surrogate for Ab1) or an antibody isotype control (13C4; data not shown) 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 96-well plates at 4 × 10 per well. 4 Target cells (MOLP8) and 1.2 × 10 5 Effector cells (NK92V) were seeded at a density of 100x100 / mL. Cells were treated with Ab2 at 0, 0.001, 0.01, 0.1, or 1 μg / mL, or IgG1 control at 1 μg / mL for 1 h at 37°C. Released calcein fluorescence 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. 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 point, exponentially growing NCI-H929 cells were labeled with calcein (AM) and incubated with Ab2 at 1 μg / mL for 30 min. NK effector cells were then resuspended and combined with labeled target NCI-H929 cells for 1 h in a humidified incubator. Ab2-mediated ADCC was quantified by measuring the levels of calcein (AM)-labeled target cells using a spectrophotometer. Increasing doses of TGF-β inhibit 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, while 1 and 10 ng / mL block ADCC by approximately 50% (Figure 4). ADCC was then assessed at two different concentrations of Ab1 (100 and 200 μg / mL). Neither the isotype control Ab (IgG4) nor Ab1 had any effect on ADCC when added to the cultures in the absence of TGF-β (Figures 5 and 6). Ab1 was shown to block 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 effect of TGF-β to the same extent. Thus, the data demonstrate that Ab1 can attenuate the immunosuppressive effect of TGF-β on NK cell-mediated ADCC.

[0168] Example 5: Neutralization of endogenous TGF-β with 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 No. 17955RF). Isolated NK cells were cultured alone or co-cultured in the presence of JJN3 cells in 6-transwell plates with or without 100 mg / ml of 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 at 37°C for 2 hours with calcein (AM) (Invitrogen catalogue no. C3100MP) for detection of 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 (Ab2) in calcein (AM)-labelled RPMI 8226 for detection of Ab2-mediated ADCC. * ) for 1 h at 37°C. Released calcein fluorescence 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 (Figure 7) and increased Ab2-mediated ADCC (Figure 8) demonstrate that the presence of Ab1 neutralized JJN3 cell-released endogenous TGF-β during the 90-h 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 are resistant to one or more other treatments that target CD38 (e.g., isatuximab or daratumumab), e.g., who are non-responsive to other treatments that target CD38 or who have 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, for example, 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 of time necessary to achieve the desired results, e.g., as determined by a physician. In some embodiments, one or both antibodies were administered via intravenous infusion. In certain embodiments, Ab2 was administered at 10 mg / kg or 16 mg / kg actual body weight for 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 pharmaceutical composition comprising an anti-CD38 antibody, or antigen-binding fragment thereof, in combination with an anti-TGF-β antibody, or antigen-binding fragment thereof, for use in treating multiple myeloma in a human patient in need thereof, comprising: The anti-CD38 antibody comprises a heavy chain CDR1 (HCDR1), a HCDR2, a HCDR3, a light chain CDR1 (LCDR1), a LCDR2, and a CDR3 each having the amino acid sequence of SEQ ID NO: 15 to 20. and LCDR3, The pharmaceutical composition, wherein the anti-TGF-β antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs:5 to 10, respectively.

2. 1. A pharmaceutical composition comprising an anti-TGF-β antibody, or an antigen-binding fragment thereof, in combination with an anti-CD38 antibody, or an antigen-binding fragment thereof, for use in treating multiple myeloma in a human patient in need thereof, comprising: The anti-TGF-β antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 (HCDR1), HCDR2, HCDR3, a light chain CDR1 (LCDR1), LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs:5-10, respectively; The pharmaceutical composition, wherein the anti-CD38 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprising the amino acid sequence of SEQ ID NO:15-20.

3. 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; and the anti-TGF-β antibody or antigen-binding fragment thereof comprises V H and V L The pharmaceutical composition according to claim 1 or 2, comprising:

4. The pharmaceutical composition of claim 1 or 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.

5. Anti-CD38 antibody is human IgG 1 The anti-TGF-β antibody contains a human IgG Fc region. 4 The pharmaceutical composition according to any one of claims 1 to 3, comprising an Fc region.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CD38 antibody and the anti-TGF-β antibody or antigen-binding fragment are administered sequentially to the patient.

7. The pharmaceutical composition or use according to any one of claims 1 to 6, wherein the treatment results in less bone destruction than treatment with an anti-CD38 antibody alone.

8. The pharmaceutical composition or use according to any one of claims 1 to 7, wherein the treatment enhances fracture healing.

9. The pharmaceutical composition or use according to any one of claims 1 to 8, wherein the treatment further comprises dexamethasone.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the multiple myeloma is refractory to treatment with Ab2 or daratumumab, or is refractory to treatment with both Ab2 and daratumumab.

11. 1. An article of manufacture comprising an anti-CD38 antibody, or antigen-binding fragment thereof, and an anti-TGF-β antibody, or antigen-binding fragment thereof, for treating multiple myeloma in a patient in need thereof, comprising: The anti-CD38 antibody: a) has HCDR1-3 and LCDR1-3 comprising the amino acid sequences of SEQ ID NOs: 15-20, respectively; b) having a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively; 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) having a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 3 and 4, respectively; or c) having a HC and a LC comprising the amino acid sequences of SEQ ID NOs: 1 and 2, respectively.

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