HK40106689A - Anti-tgf-beta antibodies and their use - Google Patents

Description

Anti-TGF-β antibodies and their uses

This invention application is a divisional application of the invention patent application filed on January 19, 2018, with application number 201880007975.3 (international application number PCT/IB2018/000088) and titled "Anti-TGF-β antibody and its use".

Cross-references to related applications

This application claims priority to U.S. Provisional Application 62/448,800 and European Application 17305061.8, both filed January 20, 2017. The disclosures of both priority applications are incorporated herein by reference in their entirety.

Technical Field

This invention relates to the field of bioengineering, and more specifically to anti-TGF-β antibodies and their uses.

Background Technology

Transforming growth factor β (TGF-β) is a cytokine that controls many key cellular functions, including proliferation, differentiation, survival, migration, and epithelial-mesenchymal transition. It regulates a variety of biological processes, such as extracellular matrix formation, wound healing, embryonic development, skeletal development, hematopoiesis, immune and inflammatory responses, and malignant transformation. Dysregulation of TGF-β leads to pathological conditions such as birth defects, cancer, chronic inflammation, and autoimmune and fibrotic diseases.

TGF-β has three known isoforms—TGF-β1, 2, and 3. All three isoforms are initially translated into a pro-peptide. Upon cleavage, the mature C-terminus remains bound to the N-terminus (called a latency-associated peptide, or LAP), forming a small latent complex (SLC) secreted from the cell. The SLC cannot bind to TGF-β receptor II (TGFβRII), preventing receptor binding. Activation by dissociation of the N- and C-termini occurs through one of several mechanisms, including proteolytic cleavage, acidic pH, or alteration of integrin structure (Connolly et al., Int J Biol Sci (2012) 8(7):964-78).

TGF-β1, 2, and 3 are pleiotropic in function and expressed in different patterns across different cell and tissue types. They exhibit similar in vitro activity, but individual knockouts in specific cell types suggest that despite their shared ability to bind the same receptor, they have different roles in vivo (Akhurst et al., Nat Rev Drug Discov (2012) 11(10):790-811). Upon binding of TGF-β to TGFβRII, the receptor's constitutive kinase activity phosphorylates and activates TGFβRI, which phosphorylates SMAD2/3, thereby allowing binding to SMAD4, localization to the nucleus, and transcription of TGF-β responsive genes. See above. In addition to this typical signaling cascade, atypical pathways transmit signals via 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, IFN, TNF, and RAS. Therefore, the final result of TGF-β signaling is crosstalk among all these signaling pathways that integrate cellular state and environment. See above.

Given the diverse functions of TGF-β, there is a need for pan-TGF-β-specific therapeutic antibodies that are safe for human patients (Bedinger et al., mAbs. (2016) 8(2):389–404). However, TGF-β is highly conserved across species. As a result, generating antibodies against human TGF-β in animals such as mice is a challenging task.

There is also a medical need for patients who currently do not receive effective treatment. For example, in the phase III Checkmate-067 study, more than 50% of patients with advanced melanoma did not have a complete or partial response to treatment with nivolumab monotherapy (Larkin et al., N Engl J Med (2015) 373:23–34; Redman et al., BMC Med (2016) 14:20–30).

Summary of the Invention

This invention provides improved monoclonal antibodies that specifically bind to human TGF-β1, TGF-β2, and TGF-β3 (i.e., pan-TGF-β specificity). These antibodies are less prone to forming haptens (i.e., dimeric complexes with one heavy chain and one light chain) during manufacturing. They also possess superior pharmacokinetic profiles, such as increased half-lives, and thus can confer improved clinical benefits to patients. The inventors have also found that TGF-β inhibition, such as that achieved by the antibodies and antigen-binding fragments of this invention, alleviates the immunosuppressive microenvironment in tumors and enhances the efficacy of immunotherapies, such as those targeting programmed cell death protein 1 (PD-1), PD-1 ligand 1 (PD-L1), and 2 (PD-L2).

On one hand, the present invention provides isolated monoclonal antibodies that specifically bind to human TGF-β1, TGF-β2, and TGF-β3, comprising heavy chain complementarity-determining regions (CDRs) 1-3 of SEQ ID NO:1 and light chain CDRs 1-3 of SEQ ID NO:2, wherein the antibody contains a human IgG4 constant region with a mutation at position 228 (EU number). In some embodiments, the mutation is a mutation that changes serine to proline (S228P). In some embodiments, the antibody comprises a heavy chain variable domain (VH) amino acid sequence corresponding to residues 1-120 of SEQ ID NO:1 and a light chain variable domain (VL) amino acid sequence corresponding to residues 1-108 of SEQ ID NO:2. In a further embodiment, the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:2. The present invention is further characterized by the F(ab') ₂ antigen-binding fragment of the above-described antibody.

In a preferred embodiment, the antibody or fragment of the present invention has an increased half-life, increased exposure, or both, compared to fresolimumab treatment. For example, the increase is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more. Exposure to a drug such as the antibody or fragment of the present invention is a function of the drug concentration in the body relative to time. The drug concentration in the body is typically expressed as the drug level in blood, plasma, or serum. The half-life and exposure (biological exposure) of the drug can be measured by well-known methods (such as those shown in Example 7 below).

The present invention also provides compositions comprising the antibodies of the present invention, wherein the compositions comprise less than 1% of a hapten. Hapten formation can be determined by purity analysis of the monoclonal antibody preparation using, for example, SDS-capillary electrophoresis or non-reducing SDS-PAGE under non-reducing conditions, followed by density determination or RP-HPLC (Angal et al., Mol Immunol (1993) 30(1):105-8; Bloom et al., Protein Science (1997) 6:407-415; Schuurman et al., (2001) 38(1):1-8; and Solanos et al., Anal Chem (2006) 78:6583-94). In some embodiments, such compositions are pharmaceutical compositions further comprising pharmaceutically acceptable excipients.

On the other hand, the present invention provides a method for inhibiting TGF-β signaling in patients (humans) in need, comprising administering a therapeutic amount of the antibody or fragment of the present invention to the patient. In some embodiments, the patient suffers from an immune-mediated disease (e.g., scleroderma), a fibrotic condition (e.g., renal fibrosis such as focal segmental glomerulosclerosis (FSGS), or pulmonary fibrosis such as idiopathic pulmonary fibrosis), or birth or bone defects (e.g., osteogenesis imperfecta). In some embodiments, the patient suffers from cancer. In some embodiments, the antibody or fragment used in the method inhibits the differentiation of CD4+ T cells into inducible regulatory T cells (iTregs). The antibody or fragment can alleviate the immunosuppressive tumor microenvironment. This action of the antibody or fragment helps to activate the immune system and enhance the efficacy of immunotherapy. The efficacy of the treatment methods described herein can be indicated by one or more of the following in patients (e.g., in the tumor tissue of patients): (1) increased levels of MIP2 and/or KC/GRO, (2) activation or infiltration of CD8+ T cells such as INF-γ positive CD8+ T cells into the tumor tissue, and (3) increased clustering of natural killer (NK) cells.

The present invention further provides a method for treating cancer in a patient (person), comprising administering to the patient (1) a therapeutically effective amount of an antibody or fragment of the present invention, and (2) a therapeutically effective amount of an immune checkpoint protein inhibitor. The two agents may be administered simultaneously (e.g., as a single composition or as separate compositions), or sequentially in any order. For example, the two agents may be administered on the same day. In some embodiments, the therapeutic agent (1) is administered to the patient prior to the therapeutic agent (2) (e.g., one or more days prior).

In some embodiments, the immune checkpoint protein is PD-1, PD-L1, or PD-L2. In a further embodiment, the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody. In a further embodiment, the anti-PD-1 antibody comprises (1) the heavy chain CDR1-3 of SEQ ID NO:5 and the light chain CDR1-3 of SEQ ID NO:6, (2) the V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO:5 and the V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO:6, or (3) the heavy chain amino acid sequence shown in SEQ ID NO:5 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:6. In one specific embodiment, the method includes administering to a cancer patient an anti-TGF-β antibody comprising the heavy chain amino acid sequence shown in SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:2, and an anti-PD-1 antibody sequence comprising the heavy chain amino acid sequence shown in SEQ ID NO:5 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:6. In some embodiments, the patient is refractory to anti-PD-1 antibody monotherapy. The patient may have advanced or metastatic melanoma, or squamous cell carcinoma of the skin.

In some regimens, patients are given anti-TGF-β antibody and anti-PD-1 antibody every 2 or 3 weeks. In some regimens, the two agents are administered at a dose of 0.01-40 (e.g., 0.02-20, 0.05-15, or 0.05-20) mg/kg body weight, respectively.

The present invention also provides a method for enhancing an immune response in patients in need, comprising administering an immune checkpoint inhibitor and the antibody or fragment of the present invention to the patient. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, for example comprising: (1) HCDR1-3 of SEQ ID NO:5 and LCDR1-3 of SEQ ID NO:6; (2) VH and VL corresponding to residues 1-117 of SEQ ID NO:5 and residues 1-107 of SEQ ID NO:6, respectively; or (3) a heavy chain having the amino acid sequence of SEQ ID NO:5 (with or without a C-terminal lysine) and a light chain having the amino acid sequence of SEQ ID NO:6.

The methods of this invention can be used to treat a variety of cancers, including but not limited to melanoma (e.g., metastatic or advanced), lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma of the skin, colorectal cancer, breast cancer, ovarian cancer, fallopian tube cancer, uterine cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), liver cancer (e.g., hepatocarcinoma), urothelial carcinoma, and kidney cancer (e.g., renal cell carcinoma). In some embodiments, the patient has a mesenchymal tumor or a mesenchymal subtype of a solid tumor. Examples of such solid tumors include those of the colon (e.g., colorectal cancer), ovaries, head and neck (e.g., head and neck squamous cell carcinoma), liver (e.g., hepatocellular carcinoma), and the urinary system.

In some implementations, cancers, including mesenchymal and neoplastic cancers, can be characterized by overexpression of one or more of ACTA2 (smooth muscle α2 actin), VIM (vimentin), MGP (matrix Gla protein), ZWINT (ZW10 interacting centromere protein), and ZEB2 (zinc finger E-box binding homeobox 2). The expression levels of such biomarkers can be determined, for example, at the mRNA or protein level, in biological samples from patients, such as tumor biopsies or circulating tumor cells.

The present invention also provides the above-described antibodies, fragments or compositions for treating the conditions described herein, and the use of the above-described antibodies, fragments or compositions in the preparation of medicaments for treating the conditions described herein.

The present invention also includes a nucleic acid expression vector encoding the heavy chain or light chain or both of the antibody of the present invention; a host cell containing the heavy chain and light chain coding sequences of the antibody; and a method for preparing the antibody using the host cell, the method comprising the steps of culturing the host cell in a suitable culture medium to allow expression of the antibody gene and subsequently harvesting the antibody.

Specifically, the present invention relates to the following technical solutions:

1. An isolated monoclonal antibody that specifically binds to human TGF-β1, TGF-β2 and TGF-β3, said antibody comprising heavy chain complementarity-determining regions (CDRs) 1-3 in SEQ ID NO:1 and light chain CDRs 1-3 in SEQ ID NO:2, wherein said antibody contains a human _IgG4 constant region having a proline at position 228 (EU number).

2. The antibody according to claim 1, wherein the antibody comprises a heavy chain variable domain ( _VH ) amino acid sequence corresponding to residues 1-120 of SEQ ID NO:1 and a light chain variable domain ( _VL ) corresponding to residues 1-108 of SEQ ID NO:2.

3. The antibody according to claim 2, wherein the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

4. An antigen-binding fragment of an antibody according to any one of items 1-3, wherein said fragment is F(ab') ₂ .

5. An antibody or fragment according to any one of items 1-4, wherein the antibody or fragment has an increased half-life or increased exposure compared to fresolimumab.

6. An antibody or fragment according to any one of items 1-5, wherein said antibody or fragment has one or more of the following properties:

a) Inhibit the differentiation of CD4 ⁺ T cells into inducible regulatory T cells (iTregs),

b) Increase CD8 ⁺ T cell proliferation

c) Increase the aggregation of natural killer (NK) cells.

d) Increase the level of MIP2, and

e) Increase the level of KC/GRO.

7. The antibody or fragment according to any one of the preceding items, which is used as a drug.

8. A composition comprising an antibody or fragment according to any one of claims 1-6, wherein the composition comprises less than 1% of a hapten or fragment.

9. A method for inhibiting TGF-β signaling in a patient in need, comprising administering to the patient a therapeutically effective amount of an antibody or fragment according to any one of claims 1-6.

10. The method according to item 9, wherein the patient has cancer.

11. The method according to item 10, wherein the cancer is selected from the group consisting of: melanoma, 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.

12. The method according to item 10 or 11, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

13. The method according to any one of items 10-12, wherein the cancer is a mesenchymal tumor.

14. The method according to any one of items 10-13, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

15. A method of treating cancer in a patient, comprising administering to the patient (1) an antibody or fragment according to any one of items 1-6, and (2) an inhibitor of an immune checkpoint protein.

16. The method according to item 15, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2.

17. The method according to item 16, wherein the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody.

18. The method according to claim 17, wherein the anti-PD-1 antibody comprises the heavy chain CDR1-3 of SEQ ID NO:5 and the light chain CDR1-3 of SEQ ID NO:6.

19. The method according to claim 17, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO:5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO:6.

20. The method according to claim 17, wherein the anti-PD-1 antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:6.

21. The method according to any one of claims 15-20, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

22. The method according to any one of items 15-21, wherein the cancer is refractory to anti-PD-1 antibody therapy.

23. The method according to any one of items 15-22, wherein the cancer is advanced or metastatic melanoma or squamous cell carcinoma of the skin.

24. The method according to any one of items 15-23, wherein the cancer is a mesenchymal subtype of a solid tumor.

25. The method according to any one of items 15-24, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

26. The method according to any one of items 15-25, wherein the cancer is selected from the group consisting of melanoma, 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.

27. The method according to any one of items 15-26, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

28. The method according to any one of items 17-27, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient on the same day.

29. The use according to any one of items 17-28, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient every two weeks.

30. The use according to any one of items 17-29, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered at a dose of 0.05-20 mg/kg body weight, respectively.

31. A method for enhancing an immune response in a subject in need, comprising administering to the patient (1) an antibody or fragment according to any one of items 1-6, and (2) an inhibitor of an immune checkpoint protein.

32. The method according to item 31, wherein the inhibitor of the checkpoint protein is an anti-PD-1 antibody.

33. The method according to claim 32, wherein the anti-PD-1 antibody comprises:

a) HCDR1-3 in SEQ ID NO:5 and LCDR1-3 in SEQ ID NO:6;

b) The V _H amino acid sequences corresponding to residues 1-117 in SEQ ID NO:5 and the V _L amino acid sequences corresponding to residues 1-107 in SEQ ID NO:6; or

c) The heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:6.

34. The method according to any one of claims 31-33, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

35. An antibody or fragment according to any one of items 1-6, for use in patients who require inhibition of TGF-β signaling.

36. The antibody or fragment provided for use according to item 35, wherein the patient has cancer.

37. The antibody or fragment for use according to item 36, wherein the cancer is selected from the group consisting of: melanoma, 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.

38. An antibody or fragment for use according to item 36 or 37, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

39. An antibody or fragment for use according to any one of items 36-38, wherein the cancer is a mesenchymal tumor.

40. An antibody or fragment for use according to any one of items 36-39, wherein the antibody or fragment alleviates an immunosuppressive tumor microenvironment.

41. An antibody or fragment according to any one of items 1-6, in combination with an inhibitor of an immune checkpoint protein for use in the treatment of cancer in patients.

42. The antibody or fragment for use according to item 41, wherein the immune checkpoint protein is PD-1, PD-L1 or PD-L2.

43. The antibody or fragment for use according to item 42, wherein the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody.

44. The antibody or fragment for use according to item 43, wherein the anti-PD-1 antibody comprises the heavy chain CDR1-3 of SEQ ID NO:5 and the light chain CDR1-3 of SEQ ID NO:6.

45. The antibody or fragment for use according to item 43, wherein the anti-PD-1 antibody comprises the V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO:5 and the V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO:6.

46. The antibody or fragment for use according to item 43, wherein the anti-PD-1 antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:6.

47. An antibody or fragment for use according to any one of items 41-46, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

48. An antibody or fragment for use according to any one of items 41-47, wherein the cancer is refractory to anti-PD-1 antibody therapy.

49. An antibody or fragment for use according to any one of items 41-48, wherein the cancer is advanced or metastatic melanoma or squamous cell carcinoma of the skin.

50. An antibody or fragment for use according to any one of items 41-49, wherein the cancer is a mesenchymal subtype of a solid tumor.

51. An antibody or fragment for use according to any one of items 41-50, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

52. An antibody or fragment for use according to any one of items 41-51, wherein the cancer is selected from the group consisting of: melanoma, 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.

53. An antibody or fragment for use according to any one of items 41-52, wherein the antibody or fragment alleviates an immunosuppressive tumor microenvironment.

54. An antibody or fragment for use according to any one of items 43-53, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient on the same day.

55. The antibody or fragment for use according to any one of items 43-54, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient every two weeks.

56. The antibody or fragment for use according to any one of items 43-55, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered at a dose of 0.05-20 mg/kg body weight, respectively.

57. An antibody or fragment according to any one of items 1-6, in combination with an inhibitor of an immune checkpoint protein, for use in patients in need of enhancing an immune response.

58. The antibody or fragment for use as described in item 57, wherein the inhibitor of the checkpoint protein is an anti-PD-1 antibody.

59. The antibody or fragment for use according to item 58, wherein the anti-PD-1 antibody comprises:

a) HCDR1-3 in SEQ ID NO:5 and LCDR1-3 in SEQ ID NO:6;

b) The V _H amino acid sequences corresponding to residues 1-117 in SEQ ID NO:5 and the V _L amino acid sequences corresponding to residues 1-107 in SEQ ID NO:6; or

c) The heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:6.

60. An antibody or fragment for use according to any one of items 57-59, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

61. Use of any antibody or fragment described in any one of items 1-6 in the preparation of a medicament for inhibiting TGF-β signaling in patients in need.

62. The use according to item 61, wherein the patient has cancer.

63. According to the use described in item 62, the cancer is selected from the group consisting of melanoma, 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.

64. The use according to item 62 or 63, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

65. The use according to any one of items 62-64, wherein the cancer is a mesenchymal tumor.

66. The use according to any one of items 62-65, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

67. Use of any antibody or fragment described in any one of items 1-6 in the preparation of a medicament for the treatment of cancer in patients in need, in combination with an inhibitor of an immune checkpoint protein.

68. The use according to item 67, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2.

69. The use according to item 68, wherein the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody.

70. The use according to claim 69, wherein the anti-PD-1 antibody comprises the heavy chain CDR1-3 of SEQ ID NO:5 and the light chain CDR1-3 of SEQ ID NO:6.

71. The use according to item 69, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO:5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO:6.

72. The use according to claim 69, wherein the anti-PD-1 antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:6.

73. The use according to any one of items 67-72, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

74. The use according to any one of items 67-73, wherein the cancer is refractory to antiPD-1 antibody therapy.

75. The use according to any one of items 67-74, wherein the cancer is advanced or metastatic melanoma or squamous cell carcinoma of the skin.

76. The use according to any one of items 67-75, wherein the cancer is a mesenchymal subtype of a solid tumor.

77. The use according to any one of items 67-76, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

78. The use according to any one of items 67-77, wherein the cancer is selected from the group consisting of melanoma, 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.

79. The use according to any one of items 67-78, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

80. The use according to any one of items 67-79, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient on the same day.

81. The use according to any one of items 68-80, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient every two weeks.

82. The use according to any one of items 69-81, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered at a dose of 0.05-20 mg/kg body weight, respectively.

83. Use of any antibody or fragment described in any one of items 1-6 in the preparation of a medicament for enhancing an immune response in patients in need, in combination with an inhibitor of an immune checkpoint protein.

84. The use according to item 83, wherein the inhibitor of the checkpoint protein is an anti-PD-1 antibody.

85. The use according to claim 84, wherein the anti-PD-1 antibody comprises:

a) HCDR1-3 in SEQ ID NO:5 and LCDR1-3 in SEQ ID NO:6;

b) The V _H amino acid sequences corresponding to residues 1-117 in SEQ ID NO:5 and the V _L amino acid sequences corresponding to residues 1-107 in SEQ ID NO:6; or

c) The heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:6.

86. The use according to any one of claims 83-85, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 and the light chain amino acid sequence shown in SEQ ID NO:2.

Attached Figure Description

Figures 1A-E illustrate the effects of Ab1, fresolimumab, and 1D11 on the proliferation of mink lung (Mv1Lu) cells treated with 1 ng/ml of human TGF-β1 (A), human TGF-β2 (B), human TGF-β3 (C), mouse TGF-β1 (D), or mouse TGF-β2 (E). Antibody concentrations are in μg/ml.

Figure 2 is a bar graph showing the effect of 50 μg/ml Ab1 on the differentiation of human inducible regulatory T cells (iTregs). The stimulation provided to the T cells was anti-CD3 and anti-CD28 antibodies plus IL-2.

Figure 3 is a bar graph showing the effect of Ab1 on the differentiation of human inducible regulatory T cells (iTregs) in human CD4+ T cell cultures treated with 2 ng/ml human TGF-β1. The stimulation provided to the T cells was anti-CD3 and anti-CD28 antibodies plus IL-2.

Figure 4 is a bar graph showing the effects of Ab1 (30 μg/ml) and human TGF-β1 (18 ng/ml) on NFATC-driven luciferase expression on Jurkat T cells after T cell stimulation and anti-PD-1 treatment.

Figure 5 shows the median tumor volume versus median absolute deviation (MAD) in the treatment groups using the C57BL/6MC38 colon mouse model. Vector: PBS. "Anti-PD-1": x-anti-mPD-1Mab (see details below). "Isotype control of Ab1": anti-HEL hIgG4.

Figure 6 is a scatter plot showing the change in tumor volume from baseline on day 27 of the treatment indicated in the C57BL/6MC38 colon mouse model. Control: PBS. "Anti-PD-1RPM114 mIgG1": x-anti-mPD-1Mab.

Figures 7A-F are graphs showing tumor volume over time in the various treatment groups using the C57BL/6MC38 colon mouse model. Each line in the graph represents one animal. “mpk”: mg/kg. “Ab1 isotype Ctrl”: anti-HEL hIgG4. αPD1: x-anti-mPD-1Mab.

Figure 8 shows the effect of Ab1 in LoVo tumor lysate on the concentration of active TGF-β1.

Figure 9A shows the serum concentrations of Ab1 and fresolimumab over time in five groups of rats administered a single dose of 5 mg/kg of any antibody. Groups (Gr.) 1–3 were administered three different batches (B1, B2, and B3) of fresolimumab. Groups 4 and 5 were administered two different batches (B1 and B2) of Ab1.

Figure 9B is a graph showing the serum concentrations of Ab1 and fresolimumab over time in monkeys given a single dose of 1 mg/kg of any of the antibodies.

Figure 9C is a graph showing the serum concentrations of Ab1 and fresolimumab over time in monkeys that were given five weekly doses of Ab1 at 1 mg/kg per dose or bi-weekly doses of fresolimumab at 1 mg/kg per dose for the specified study duration.

Figure 9D is a graph showing the serum concentrations of Ab1 and fresolimumab over time in monkeys given a single dose of 10 mg/kg of any of the antibodies.

Figure 9E is a graph showing the serum concentrations of Ab1 and fresolimumab over time in monkeys that were given five weekly doses of Ab1 at 10 mg/kg each or bi-weekly doses of fresolimumab at 10 mg/kg each for the specified study duration.

Figure 10A shows the changes in TGF-β1 levels in MC38 tumors after treatment with Ab1 (+/- anti-PD1).

Figure 10B shows the changes in MIP-2 levels in MC38 tumors after treatment with Ab1 (+/- anti-PD1).

Figure 10C shows the changes in KC/GRO levels in MC38 tumors after treatment with Ab1 (+/- anti-PD1).

Figure 11A shows the quantification of CellTrace Violet staining and IFN-γ staining in CD8 ^pos cells.

Figure 11B shows that Ab1 restored proliferation and IFN-γ production in TGFβ-treated CD8+ T cells.

Figure 12A is a graph showing the relative abundance (log2-transformed) of CD8+ T cells across the entire syngeneic mouse tumor models of colon cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma, and renal cancer.

Figure 12B is a diagram showing the activation of the TGFβ pathway across an outline of syngeneic mouse tumor models of colon cancer, leukemia, lung cancer, lymphoma, breast cancer, melanoma, mesothelioma, and renal cancer.

Invention Details

The present invention is characterized by improved pan-TGF-β specific monoclonal antibodies that are less prone to hapten formation and also possess superior pharmacokinetic profiles, such as higher in vivo exposure than existing known antibodies. The antibodies of the present invention are collectively referred to as "Ab1 and related antibodies" and share common structural features, namely, they have heavy chain CDRs (HCDRs) 1-3 in SEQ ID NO:1 and light chain CDRs (LCDRs) 1-3 in SEQ ID NO:2, and have a human IgG4 constant region in which residue 228 (EU number) in the hinge region has been mutated from serine to proline. P228 is indicated by a box and bold text in the sequence of SEQ ID NO:1 shown below.

When unglycosylated, antibody Ab1 has an estimated molecular weight of 144 kDa. Its heavy and light (chain) amino acid sequences are SEQ ID NO: 1 and 2, respectively. These sequences are shown below. Variable domains are in italics. CDRs are shown in boxes. Glycosylation sites in the heavy chain constant domain are indicated in bold (N297).

In some embodiments, the antibodies of the present invention, such as anti-TGFβ antibodies, do not have a C-terminal lysine in the heavy chain. The C-terminal lysine can be removed during preparation or by recombination techniques (i.e., the coding sequence of the heavy chain does not include a codon for the C-terminal lysine). Therefore, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO:1 that does not have a C-terminal lysine are also contemplated within the present invention.

Ab1 and its associated antibodies specifically bind to human TGF-β1, -β2, and -β3. "Specificity" means that the binding, as determined by, for example, surface plasmon resonance (see, for example, Example 1 below) or bio-layer interference, has _{a KD} of less than ^10⁻⁷ M, e.g., less than ^10⁻⁸ M (e.g., 1-5 nM). Ab1 and its associated antibodies may also have strong TGF-β neutralizing potency as determined in a mink lung epithelial cell assay (see, for example, Example 2 below), or an EC50 of approximately 0.05-1 μg/ml as determined in an A549 cell IL-11 induction assay (see, for example, Example 6 in PCT Publication WO 2006/086469, the disclosure of which is incorporated herein by reference in its entirety).

The antigen-binding and neutralizing properties of Ab1 and the associated antibody are comparable to those of the previous anti-TGF-β antibody fresolimumab (germinated IgG ₄ PET1073G12 antibody described in WO 2006/086469). The heavy and light chain sequences of fresolimumab, including the leader sequence, are shown in SEQ ID NO:3 and 4, respectively. As can be seen in SEQ ID NO:3, fresolimumab does not have proline at position 228 (EU number, which corresponds to actual position 247 in SEQ ID NO:3). Ab1 and the associated antibody have several improved features compared to fresolimumab.

During manufacturing, fresolimumab can form up to 6-18% hemiantibodies (i.e., dimers with one heavy chain and one light chain, rather than tetramers with two heavy chains complexed with two light chains) under non-reducing denaturing conditions. In contrast, Ab1 produces significantly reduced hemiantibodies (<1%). Therefore, Ab1 and related antibodies result in a purer drug product during manufacturing.

Furthermore, Ab1 and related antibodies may exhibit improved pharmacokinetic (PK) profiles compared to fresolimumab. They may exhibit linear PK behavior with a significantly longer half-life and lower elimination rate than fresolimumab, resulting in approximately 1.7-fold higher in vivo exposure. For example, in rats, Ab1 has been shown to have a mean half-life of 7.1 days compared to 4.3 days for fresolimumab, and an elimination rate (CL) of 0.30 ml/hr/kg compared to 0.51 ml/hr/kg for fresolimumab (Example 7, hereinafter). In cynomolgus monkeys, Ab1 has been shown to have a mean half-life of 13 days compared to 4.5 days for fresolimumab, and an elimination rate (CL) of 0.40 ml/hr/kg compared to 0.66 ml/hr/kg for fresolimumab. Ibid. These improved PK properties suggest that Ab1 and the associated antibody can be administered to patients at lower doses and/or lower frequencies than fresolimumab to achieve the same or better clinical outcomes, while causing fewer adverse side effects and fewer anti-drug antibody responses, thus allowing for longer treatment durations where necessary.

Furthermore, during toxicological studies of fresolimumab in non-human primates, a correlation between drug exposure and adverse events such as anemia was observed. However, in similar studies with Ab1 at comparable or even higher exposure levels, no such events were observed.

Without being bound by theory, we assume that mutations in residue 228 of the heavy chain of Ab1 and related antibodies lead to increased stability and improved PK and toxicological profiles.

The constant domains of Ab1 and related antibodies can be further modified as needed, for example at Kabat residue L248 (e.g., by introducing the mutation L248E) to reduce any unwanted effector functions of the molecule.

As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetrameric protein comprising two heavy (H) chains (approximately 50-70 kDa) and two light (L) chains (approximately 25 kDa) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable domain ( _VH ) and a heavy chain constant domain ( _CH ). Each light chain consists of a light chain variable domain ( _VL ) and a light chain constant domain ( _CL ). _{The VH} and _VL domains can be further subdivided into hypervariable regions called "complementarity-determining regions" (CDRs), in between which are more conserved regions called "framework regions" (FRs). Each _VH or _VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Amino acids can be assigned to each region according to the definitions (Lefranc et al., Dev CompImmunol 27(1):55-77(2003); or Kabat's definition, 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).

The term "human antibody" refers to an antibody in which both the variable and constant regions are derived from human sequences. This term encompasses antibodies with sequences derived from human genes, but those sequences have been modified, for example, to reduce immunogenicity, increase affinity, and increase stability. The term also includes antibodies recombinantly generated in non-human cells, which can confer glycosylations atypical for human cells.

The term "chimeric antibody" refers to an antibody that contains sequences from two different animal species. For example, a chimeric antibody may contain _VH and _VL of a mouse antibody (i.e., an antibody encoded by a mouse antibody gene, such as an antibody obtained from an _{immunized mouse} using hybridoma technology), which are linked to constant regions of an antibody from another species (e.g., a human, rabbit, or rat).

The term "antigen-binding fragment" in the context of an antibody refers to an antibody fragment that retains the ability to specifically bind to an antigen. In some embodiments, the antigen-binding fragment of the present invention is an F(ab') ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region (Fab is a monovalent antibody fragment composed of _VL , _VH , _CL , and _CH1 domains). In some embodiments, the antigen-binding fragment of the present invention may also comprise a _CH2 or _CH3 domain.

The antibody and antigen-binding fragments described herein can be isolated. The terms “isolated protein,” “isolated polypeptide,” or “isolated antibody” refer to a protein, polypeptide, or antibody that, by virtue of its origin or derived source, (1) is not associated with the naturally associated component that accompanies it in its native state, (2) is substantially free of other proteins from the same species, (3) is expressed by cells from a different species, or (4) is not present in nature. Thus, polypeptides synthesized chemically or in cellular systems different from the cells of their natural origin will be “isolated” from their naturally associated components. Isolation can also render proteins substantially free of their naturally associated components by using protein purification techniques known in the art.

Uses of I.Ab1 and related antibodies

TGF-β receptors are widely expressed on immune cells, leading to a broad role for TGF-β in both the innate and adaptive immune systems. TGF-β is associated with many disease conditions, such as birth defects, cancer, chronic inflammation, autoimmune diseases, and fibrotic disorders. Therapeutic doses of Ab1 or related antibodies can be used to treat these conditions. A “therapeuticly effective” dose refers to the amount of Ab1, related antibody, or another therapeutic agent mentioned herein that reduces one or more symptoms of the treated condition. This dose may vary depending on the condition being treated or the patient and can be determined by a healthcare professional using well-established principles.

In some embodiments, Ab1 or the associated antibody may be administered at 40, 20, or 15 mg/kg or less (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg/kg). In some other embodiments, the dose may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 mg/kg. Dosing frequency may be, for example, daily, every two days, every three days, every four days, or every five days, weekly, every two weeks, or every three weeks, monthly, or every two months. The antibody may be administered intravenously (e.g., intravenous infusion over 0.5–8 hours), subcutaneously, topically, or by any other route of administration suitable for the condition and pharmaceutical formulation.

Ab1 and its associated antibodies are derived from human antibody genes and therefore exhibit low immunogenicity in humans. Toxicological studies of Ab1 are detailed in Example 8 below. Certain cardiac and pulmonary side effects were observed in rats. Therefore, adverse events in patients treated with Ab1 or its associated antibodies should be monitored.

In some embodiments, the efficacy of the antibody of the present invention may be indicated by one or more of the following in the patient (e.g., in the affected tissue, such as the patient’s tumor tissue): (1) a decrease in TGF-β levels or activity, (2) an increase in MIP2 and/or KC/GRO levels, (3) activation or infiltration of CD8+ T cells, such as INF-γ positive CD8+ T cells, into the tumor tissue, and (4) an increase in the aggregation of natural killer (NK) cells.

A. Non-tumor disease status

Conditions that can be treated with Ab1 and related antibodies include, but are not limited to, bone defects (e.g., osteogenesis imperfecta), glomerulonephritis, neurogenic or skin scarring, lung or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced fibrosis, liver fibrosis, myelofibrosis, scleroderma, immune-mediated diseases (including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Sjogren’s syndrome, Berger’s disease, and transplant rejection), and Dupuytren’s contracture.

They can also be used to treat, prevent, and reduce the risk of developing renal insufficiency, including but not limited to focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (type I and II), radiation nephropathy, obstructive nephropathy, diffuse systemic sclerosis, hereditary kidney diseases (e.g., polycystic kidney disease, medullary sponge kidney, horseshoe kidney), glomerulonephritis, nephrosclerosis, nephrocalcinosis, systemic or glomerular hypertension, tubulointerstitial nephropathy, renal tubular acidosis, renal tuberculosis, and renal infarction. In particular, they are useful in combination with antagonists of the renin-angiotensin-aldosterone system, including but not limited to: renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, Ang II receptor antagonists (also known as "Ang II receptor blockers"), and aldosterone antagonists. See, for example, WO 2004/098637, the disclosure of which is incorporated herein by reference in its entirety.

Ab1 and related antibodies can be used to treat diseases and conditions associated with ECM deposition, such as systemic sclerosis, postoperative adhesions, keloids and proliferative scarring, proliferative vitreoretinopathy, glaucoma drainage surgery, corneal injury, cataracts, Peroni disease, adult respiratory distress syndrome, cirrhosis, scarring after myocardial infarction, restenosis after angioplasty, scarring after subarachnoid hemorrhage, fibrosis after laminectomy, fibrosis after tendon and other repairs, biliary cirrhosis (including sclerosing cholangitis), pericarditis, pleurisy, tracheotomy, penetrating CNS injury, eosinophilic myositis syndrome, vascular restenosis, venous occlusive disease, pancreatitis, and psoriatic arthritis.

Ab1 and related antibodies are further used in conditions where promoting epithelial regeneration is beneficial. These conditions include, but are not limited to, skin diseases such as venous ulcers, ischemic ulcers (pressure sores), diabetic ulcers, transplant sites, transplant donor sites, abrasions and burns; bronchial epithelial diseases such as asthma and ARDS; intestinal epithelial diseases such as mucositis associated with cytotoxic therapy, esophageal ulcers (reflexia), gastroesophageal reflux disease, gastric ulcers, and small and large bowel injuries (inflammatory bowel disease).

Another use of Ab1 and related antibodies is in conditions where endothelial cell proliferation is desired, such as stabilizing atherosclerotic plaques and promoting the healing of vascular anastomoses, or in conditions where smooth muscle cell proliferation is desired, such as arterial disease, restenosis, and asthma.

Ab1 and related antibodies can also be used to enhance the immune response to macrophage-mediated infections, such as those caused by *Leishmania spp.*, *Trypanosorna cruzi*, *Mycobacterium tuberculosis*, and *Mycobacterium leprae*, as well as the protozoa *Toxoplasma gondii*, and the fungi *Histoplasma capsulatum*, *Candida albicans*, *Candida parapsilosis*, and *Cryptococcus neoformans*. They can also be used to reduce immunosuppression caused by conditions such as cancer, AIDS, or granulomatous diseases.

Ab1 and related antibodies can also be used to prevent and/or treat ophthalmic conditions such as glaucoma and scarring after trabeculectomy.

B. Cancer Disease Status

TGF-β regulates several biological processes, including cell proliferation, epithelial-mesenchymal transition (EMT), matrix remodeling, angiogenesis, and immune function. Each of these processes contributes to tumor progression. The widespread adverse effects of TGF-β in cancer patients across indications are also suggested by its elevation both within the tumor microenvironment and systemically. See, for example, Kadam et al., Mol. Biomark. Diagn. (2013) 4(3). Studies have shown that in malignant states, TGF-β can induce EMT, and the resulting mesenchymal phenotype leads to increased cell migration and invasion.

Ab1 and related antibodies can be used to treat hyperproliferative diseases such as cancers, including but not limited to skin cancer (e.g., melanoma, including unresectable or metastatic melanoma, squamous cell carcinoma of the skin, and keratoacanthoma), lung cancer (e.g., non-small cell lung cancer), esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), primary peritoneal cancer, bladder cancer, renal cancer (e.g., kidney cell carcinoma), urothelial carcinoma, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, prostate cancer, testicular cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), brain cancer, glioblastoma, glioma, mesothelioma, leukemia, and lymphoma.

In some implementations, Ab1 and related antibodies can be used to treat cancer in patients who have failed or are expected to fail prior treatment with anti-PD-1, anti-PD-L1, or anti-PD-L2 agents, i.e., patients who are non-responders or are expected to be non-responders to anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy. In some implementations, Ab1 and related antibodies can be used to treat cancer in patients who have relapsed from previous anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy. As used herein, the term “expected” means, without the administration of treatment, that a person skilled in the medical field, based on his/her general medical knowledge and the patient’s specific circumstances, can foresee whether the patient will be a responder or a non-responder, and whether the therapy will fail or will not be effective.

In some implementations, the cancer is a mesenchymal subtype of a solid tumor, including but not limited to mesenchymal colorectal cancer, mesenchymal ovarian cancer, mesenchymal lung cancer, mesenchymal head cancer, and mesenchymal neck cancer. Epithelial-mesenchymal transition (EMT) promotes cell migration and invasion by downregulating epithelial cell genes and enhancing mesenchymal gene expression. EMT is a marker of tumor progression and invasion. Up to one-quarter of colorectal and ovarian cancers are mesenchymal. Therefore, by inhibiting TGF-β and its induction of EMT, Ab1 or related antibodies can be used to treat mesenchymal solid tumors. Mesenchymal subtypes of solid tumors can be identified by a number of genetic markers and pathological tests. Markers include ACTA2, VIM, MGP, ZEB2, and ZWINT, which can be detected by qRT-PCR or immunohistochemistry. Such markers can be used to select patients for anti-TGF-β monotherapy or combination therapy according to the present invention.

In some implementations, Ab1 and related antibodies can be used to treat patients with advanced solid tumors.

Ab1 and related antibodies can also be used to treat hematopoietic disorders or malignancies such as multiple myeloma, myelodysplastic syndrome (MDS), Hodgkin lymphoma, non-Hodgkin lymphoma, leukemia, and various sarcomas such as Kaposi's sarcoma.

Ab1 and related antibodies can also be used to inhibit cyclosporine-mediated malignant tumor or cancer progression (e.g., metastasis).

It is understandable, then, that in the context of cancer treatment, “treatment” includes any medical intervention that causes cancer growth to slow, cancer progression or recurrence to be delayed, or cancer metastasis to be reduced, or cancer to achieve partial remission in order to extend the patient’s life expectancy.

C. Combination therapy in oncology

The level of cytotoxic T cell infiltration in cancer has been observed to be associated with favorable clinical outcomes (Fridman et al., Nat Rev Cancer (2012) 12(4):298–306; and Galon et al., Immunity (2013) 39(1):11–26). Additionally, helper T cells (CD4+ _TH1 ) and their produced cytokines (e.g., INF-γ) are also generally associated with positive patient outcomes. Conversely, the presence of Treg cells has been shown to be associated with poor patient prognosis (Fridman, see above).

TGF-β inhibits almost all aspects of the anti-tumor immune response. This cytokine promotes iTreg differentiation and reduces the proliferation and invasion of cytotoxic (CD8 ⁺ ) cells. As mentioned above, inhibiting TGF-β by Ab1 or related antibodies will alleviate the immunosuppressive tumor microenvironment, thereby leading to positive outcomes for cancer patients.

Furthermore, the inventors have discovered that by mitigating the immunosuppressive tumor microenvironment, Ab1 and related antibodies can allow checkpoint modulators (such as anti-PD-1 antibodies) to better induce immune responses. Therefore, more patients can benefit from immunotherapies such as anti-PD-1, anti-PD-L1, or anti-PD-L2 treatment.

With or without therapeutic agents targeting immune checkpoint molecules, Ab1 and related antibodies can also be used in combination with other cancer therapies such as chemotherapy (e.g., platinum- or taxane-based therapies), radiation therapy, and therapies targeting cancer antigens or oncogens.

Cancers that can be treated with combinations involving Ab1 or related antibodies and immune checkpoint inhibitors such as anti-PD-1 antibodies include those listed in the previous section.

In some implementations, the cancer is refractory to prior anti-PD-1, anti-PD-L1, or anti-PD-L2 therapy, such as advanced or metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and Hodgkin's lymphoma. Refractory patients are those whose disease has progressed, for example, confirmed by the absence of any response by radiological methods within 12 weeks of starting treatment.

In some implementations, Ab1 or related antibodies can be used in combination with another cancer therapy, such as anti-PD-1 therapy, to treat mesenchymal cancers, such as colorectal cancer, non-small cell lung cancer, ovarian cancer, bladder cancer, head and neck squamous cell carcinoma, renal cell carcinoma, hepatocellular carcinoma, and cutaneous squamous cell carcinoma. See also the discussion above.

Examples of anti-PD-1 antibodies are nivolumab, pembrolizumab, pidilizumab, MEDI0608 (formerly AMP-514; see, for example, WO2012/145493 and U.S. Patent 9,205,148), PDR001 (see, for example, WO2015/112900), PF-06801591 (see, for example, WO 2016/092419), and BGB-A317 (see, for example, WO 2015/035606). In some implementations, the anti-PD-1 antibody includes those disclosed in WO 2015/112800 (such as those referred to as H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH90...). Those of H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P, and H4xH9008P, and those referred to as H4H7798N, H4H7795N2, H4H9008P, and H4H9048P2 in Table 3 of the PCT disclosure. The disclosure of WO2015/112800 is incorporated herein by reference in its entirety.

For example, antibodies and related antibodies disclosed in WO 2015/112800, including antibody-antigen binding fragments having the CDR, _VH , and _VL sequences or heavy and light chain sequences disclosed in that PCT disclosure, and antibody-antigen binding fragments binding to the same PD-1 epitope as the antibody disclosed in that PCT disclosure, can be used in combination with the Ab1 or related antibodies of the present invention for the treatment of cancer. In related embodiments, a useful anti-PD-1 antibody may comprise the heavy and light chain amino acid sequences shown below as SEQ ID NO:5 and 6, respectively; the _VH and _VL sequences (shown in italics) in SEQ ID NO:5 and 6, or one or more (e.g., all six) CDRs (shown in boxes) in SEQ ID NO:5 and 6.

In some embodiments, the antibodies of the present invention, such as anti-PD-1 antibodies, do not have a C-terminal lysine in the heavy chain. The C-terminal lysine can be removed during manufacturing or by recombination techniques (i.e., the coding sequence of the heavy chain does not include a codon for the C-terminal lysine). Thus, antibodies comprising the heavy chain amino acid sequence of SEQ ID NO:5 without the C-terminal lysine are also contemplated within the present invention.

In some embodiments, the anti-TGF-β antibody or fragment of the present invention may also be used in combination with antibodies against immunomodulatory antigens such as PD-L1 and CTLA-4. Exemplary anti-PD-L1 antibodies are atezolizumab, avelumab, durvalumab, LY3300054, and BMS-936559. Exemplary anti-CTLA-4 antibodies are ipilimumab or tremelimumab.

D. Biomarkers of therapeutic efficacy

The efficacy of Ab1 and related antibodies can be determined through biomarkers or target occupancy. For example, in tumor tissue, target occupancy can be determined by assessing the level of active TGF-β in biopsy tissue using the Meso Scale Discovery (MSD) assay. In blood, target binding can be determined by assessing the effect of reduced circulating TGF-β on peripheral blood monocytes such as lymphocytes (T cells, B cells, NK cells) and monocytes. For example, CD45 ⁺ RO ⁺ CCR7 ⁺ CD28 ⁺ Ki67 ⁺ can be used as a marker in flow cytometry to assess increased proliferation of circulating CD8+ T cells. Activation of circulating NK cells can be assessed using CD3 ^- CD56 ^high/dim CD16 ⁺ or CD137 ⁺ as markers in flow cytometry. Furthermore, Ki-67, PD-1, and ICOS can be used as PD markers associated with T cell activation.

Changes in infiltrating immune cells and immune markers can be assessed using multiplex immunohistochemical (IHC) assays, such as those from the NeoGenomics platform, thereby allowing for the determination of immune modulation following treatment with Ab1 or related antibodies. Specifically, NeoGenomics' MultiOmyx TIL Panel stains a set of immune markers, allowing for the quantitative determination of the density and localization of various immune cells. Immune markers can indicate iTreg differentiation; CD8+ T cell infiltration and proliferation; and IFNγ production by CD8 ⁺ T cells. Ab1 has been shown to inhibit CD4 ⁺ T cell differentiation into iTregs (see, for example, Example 3 below) and increase CD8 ⁺ T cell proliferation and IFNγ production (as shown in the mixed lymphocyte reaction assay; data not shown). Therefore, the therapeutic efficacy of Ab1 or related antibodies can be indicated by the inhibition of iTregs, the induction of CD8 ⁺ T cell proliferation and infiltration into tumors or other diseased tissues, increased IFNγ production, and/or an increased ratio of CD8 ⁺ T cells to Treg cells. Immunomodulation following Ab1 or related antibody treatment can also be measured in peripheral blood by quantitative counting of CD8 ⁺ T cells, Treg cells, NK cells, and other immune cells based on methylation-PCR. Clinically, therapeutic efficacy can manifest as delayed or reversed disease progression, such as tumor progression.

II. Methods for preparing antibodies

Ab1 and related antibodies, as well as antibodies targeting other common targets such as PD-1, PD-L1, or PD-L2, can be prepared using methods well-established in the art. DNA sequences encoding the antibody heavy and light chains can be inserted into an expression vector, allowing the gene to be operatively linked to necessary expression control sequences, such as transcription and translation control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, colloids, YACs, EBV-derived episomes, etc. The antibody light chain coding sequence and the antibody heavy chain coding sequence can be inserted into separate vectors and can be operatively linked to the same or different expression control sequences (e.g., promoters). In one embodiment, both coding sequences are inserted into the same expression vector and can be operatively 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). Antibody coding sequences can be inserted into expression vectors using standard methods (e.g., linking antibody gene fragments to complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present).

In addition to antibody chain genes, recombinant expression vectors can carry regulatory sequences that control antibody chain gene expression in host cells. Examples of regulatory sequences used for expression in mammalian host cells include viral elements that direct high-level protein expression in mammalian cells, derived from promoters and/or enhancers such as: retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian virus 40 (SV40) (e.g., SV40 promoter/enhancer), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), polyomaviruses, and strong mammalian promoters such as innate immunoglobulin and actin promoters.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may carry additional sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selection marker genes. For example, selection marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells in which the vector has been introduced. Selection marker genes may include a dihydrofolate reductase (DHFR) gene (for DHFR-host cells with methotrexate selection/amplification), a neo gene (for G418 selection), and a glutamate synthase gene.

The expression vector encoding the antibody of the present invention is introduced into host cells for expression. The host cells are cultured under conditions suitable for antibody expression, and then the antibody is harvested and isolated. Host cells include mammalian, plant, bacterial, or yeast host cells. Mammalian cell lines that can be used as expression hosts are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, young hamster kidney (BHK) cells, African green monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Cell lines can be selected based on their expression levels. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells.

Furthermore, many known techniques can be used to enhance antibody expression. For example, the glutamine synthase gene expression system (GS system) is a commonly used method to enhance expression under certain conditions.

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

III. Pharmaceutical Composition

The antibodies of the present invention can be formulated for suitable storage stability. For example, antibodies can be lyophilized, stored, or reconstituted for use using pharmaceutically acceptable excipients. For combination therapies, two or more therapeutic agents, such as antibodies, can be co-formulated, for example, mixed, and provided as a single composition.

This document uses the term "excipient" to describe any component other than the compounds of this invention. The selection of excipients depends largely on factors such as the specific administration method, the effect of the excipient on solubility and stability, and the nature of the dosage form. "Pharmaceutically acceptable excipients" include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, and combinations thereof. In some cases, isotonic agents, such as sugars, polyols like mannitol, sorbitol, or sodium chloride, are included in the composition. Other examples of pharmaceutically acceptable substances are wetting agents or small amounts of excipients, such as wetting agents or emulsifiers, preservatives, or buffers, which increase the shelf life or efficacy of antibodies.

The pharmaceutical compositions of the present invention can be prepared, packaged, or sold in bulk as a single unit dose or as multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject or a convenient portion of that dose, such as half or one-third of the dose.

The pharmaceutical compositions of the present invention are generally suitable for parenteral administration. As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physically destroying the subject’s tissue and administering the pharmaceutical composition through said rupture in the tissue, thus generally resulting in direct administration into the bloodstream, muscle, or into internal organs. Therefore, parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection, administration through a surgical incision, administration through a tissue-penetrating non-surgical wound, etc. In particular, contemplated parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, and intrasynovial injection or infusion; and renal dialysis infusion techniques. Regional perfusion is also contemplated. Preferred embodiments may include intravenous and subcutaneous routes.

Formulations of pharmaceutical compositions intended for parenteral administration typically comprise an active ingredient in combination with a pharmaceutically acceptable carrier (e.g., sterile water or sterile isotonic saline). Such formulations may be prepared, packaged, or sold in bolus administration or continuous administration forms. Injectable formulations may be prepared, packaged, or sold in unit dose forms, such as in ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, etc., in oily or aqueous carriers. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable carrier (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (e.g., having a pH of 3-9), but for some applications, they may more suitably be formulated as sterile non-aqueous solutions or used as a dry form with a suitable carrier such as sterile pyrogen-free water. Exemplary parenteral formulations include solutions or suspensions in sterile aqueous solutions, such as aqueous propylene glycol or dextran solutions. This dosage form may be appropriately buffered if desired. Other available parenteral formulations include those containing the active ingredient in microcrystalline form or lipid preparations. Formulations for parenteral administration may be formulated for immediate and/or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release, and programmed release.

IV. Exemplary Implementation

Further specific embodiments of the present invention are described below.

1. An isolated monoclonal antibody that specifically binds to human TGF-β1, TGF-β2 and TGF-β3, said antibody comprising heavy chain complementarity-determining regions (CDRs) 1-3 in SEQ ID NO:1 and light chain CDRs 1-3 in SEQ ID NO:2, wherein said antibody contains a human _IgG4 constant region having a proline at position 228 (EU number).

2. The antibody of embodiment 1, wherein the antibody comprises a heavy chain variable domain ( _VH ) amino acid sequence corresponding to residues 1-120 of SEQ ID NO:1 and a light chain variable domain ( _VL ) corresponding to residues 1-108 of SEQ ID NO:2.

3. The antibody of embodiment 2, wherein the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 (with or without C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:2.

4. An antigen-binding fragment of the antibody as described in embodiment 3, wherein the fragment is F(ab') ₂ .

5. An antibody or fragment of any of embodiments 1-4, wherein the antibody or fragment has an increased half-life or increased exposure compared to fresolimumab.

6. An antibody or fragment as described in any one of embodiments 1-5, wherein said antibody or fragment has one or more of the following properties:

a) Inhibit the differentiation of CD4 ⁺ T cells into inducible regulatory T cells (iTregs),

b) Increase CD8 ⁺ T cell proliferation

c) Increase the aggregation of natural killer (NK) cells.

d) Increase the level of MIP2, and

e) Increase the level of KC/GRO.

7. A composition comprising an antibody or fragment of any one of embodiments 1-6, wherein the composition comprises less than 1% of a hapten.

8. An antibody or fragment thereof, as described in any of the implementation schemes 1-6, may be used as a drug.

9. A method for inhibiting TGF-β signaling in a patient in need, comprising administering to the patient a therapeutically effective amount of an antibody or fragment as described in any one of embodiments 1-6.

10. The method of embodiment 9, wherein the patient has cancer.

11. The method of embodiment 10, wherein the cancer is selected from the group consisting of: melanoma, 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.

12. The method of embodiment 10 or 11, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

13. The method of any one of embodiments 10-12, wherein the cancer is a mesenchymal tumor.

14. The method of any one of embodiments 10-13, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

15. A method of treating cancer in a patient, comprising administering to the patient (1) an antibody or fragment as described in any one of embodiments 1-6, and (2) an inhibitor of an immune checkpoint protein.

16. The method of embodiment 15, wherein the immune checkpoint protein is PD-1, PD-L1, or PD-L2.

17. The method of embodiment 16, wherein the inhibitor of the immune checkpoint protein is an anti-PD-1 antibody.

18. The method of embodiment 17, wherein the anti-PD-1 antibody comprises the heavy chain CDR1-3 of SEQ ID NO:5 and the light chain CDR1-3 of SEQ ID NO:6.

19. The method of embodiment 17, wherein the anti-PD-1 antibody comprises a V _H amino acid sequence corresponding to residues 1-117 of SEQ ID NO:5 and a V _L amino acid sequence corresponding to residues 1-107 of SEQ ID NO:6.

20. The method of embodiment 17, wherein the anti-PD-1 antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:5 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:6.

21. The method of any one of embodiments 15-20, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:2.

22. The method of any one of embodiments 15-21, wherein the cancer is refractory to anti-PD-1 antibody therapy.

23. The method of any one of embodiments 15-22, wherein the cancer is advanced or metastatic melanoma or squamous cell carcinoma of the skin.

24. The method of any one of embodiments 15-23, wherein the cancer is a mesenchymal subtype of a solid tumor.

25. The method of any one of embodiments 15-24, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

26. The method of any one of embodiments 15-25, wherein the cancer is selected from the group consisting of: melanoma, 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.

27. The method of any one of embodiments 15-26, wherein the antibody or fragment alleviates the immunosuppressive tumor microenvironment.

28. The method of any one of embodiments 17-27, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient on the same day.

29. As used in any of embodiments 17-28, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered to the patient every two weeks.

30. As used in any of embodiments 17-29, wherein the anti-TGF-β antibody and the anti-PD-1 antibody are administered at a dose of 0.05-20 mg/kg body weight, respectively.

31. A method for enhancing an immune response in a subject in need, comprising administering an immune checkpoint inhibitor and an antibody or fragment as described in any one of embodiments 1-6 to the patient.

32. The method of embodiment 31, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

33. The method of embodiment 32, wherein the anti-PD-1 antibody comprises:

a) HCDR1-3 in SEQ ID NO:5 and LCDR1-3 in SEQ ID NO:6;

b) V _H and V _L corresponding to residues 1-117 in SEQ ID NO:5 and residues 1-107 in SEQ ID NO:6, respectively; or

c) A heavy chain (with or without a C-terminal lysine) having the amino acid sequence shown in SEQ ID NO:5 and a light chain having the amino acid sequence shown in SEQ ID NO:6.

34. The method of any one of embodiments 31-33, wherein the anti-TGF-β antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO:1 (with or without a C-terminal lysine) and the light chain amino acid sequence shown in SEQ ID NO:2.

35. The method of any one of embodiments 31-34, wherein the patient has cancer.

36. The method of embodiment 35, wherein the patient is refractory to prior treatment with the immune checkpoint inhibitor and/or has a mesenchymal subtype of solid tumor.

37. The method of embodiment 35 or 36, wherein the cancer is selected from the group consisting of: melanoma, 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.

38. The method of any one of embodiments 35-37, wherein the cancer is characterized by overexpression of one or more of ACTA2, VIM, MGP and ZWINT.

39. The method of any one of embodiments 35-38, wherein the antibody or fragment alleviates the immunosuppressive tumor environment.

40. An antibody or fragment of any of the embodiments 1-6 may be used in any of the above methods to treat patients.

41. The use of an antibody or fragment of any of embodiments 1-6 for the preparation of a medicament for treating a patient in any of the above methods.

42. An isolated nucleic acid molecule comprising a heavy chain, light chain, or both of an antibody or fragment of any one of embodiments 1-6.

43. An expression vector comprising the isolated nucleic acid molecule of embodiment 42.

44. A host cell comprising the expression vector of embodiment 43.

45. A method for generating an antibody or antigen-binding fragment according to any one of embodiments 1-6, said method comprising:

A host cell is provided, the host cell containing first and second nucleotide sequences encoding a heavy chain and a light chain, respectively, that encode the antibody or antigen-binding fragment.

The host cells are grown under conditions that allow the production of the antibody or antigen-binding fragment, and

The antibody or antigen-binding fragment is recovered.

46. A method for producing a pharmaceutical composition, comprising:

Provide an antibody or antigen-binding fragment of any one of implementation schemes 1-6, and

The antibody or antigen-binding fragment is mixed with a pharmaceutically acceptable excipient.

47. An article or kit comprising an antibody or antigen-binding fragment of any one of embodiments 1-6 and another therapeutic agent.

48. An article or kit as described in embodiment 47, wherein the other therapeutic agent is an immune checkpoint inhibitor as described herein.

The invention will be further described in the following embodiments, which do not limit the scope of the invention as set forth in the claims.

Example

To better understand the present invention, the following embodiments are described. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1: TGF-β-binding properties of Ab1

The affinity of Ab1 for all human and rodent TGF-β isoforms was determined using surface plasmon resonance on a Biacore T200 biosensor instrument (GE Healthcare) with a series of dextran-coated carboxymethylated (CM5) S-chips. Ab1 was injected into immobilized recombinant TGF-β at a series of concentrations (1.11, 3.33, 10, and 30 nM) to measure binding interactions in real time. TGF-β homodimers were immobilized at low densities to reduce affinity effects. Injections were performed in triplicate, and binding measurements were repeated three times. Data from kinetic experiments were processed using Biacore T200 Biaevaluation v2.0 software. The resulting sensor maps were zeroed, aligned, double-referenced, and cropped using a 1:1 binding model for curve fitting analysis to determine the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant ( _KD ).

The recombinant proteins were either internally generated (human TGF-β1, 2, and 3) or obtained from R&D Systems (mouse TGF-β1 and 2). Table 1 below shows the amino acid sequence homology of the three active TGF-β isoforms among rhesus monkeys, mice, or rats and humans (homology is reported as a percentage of conserved amino acids relative to total amino acids).

Table 1. Homology of TGF-β active isotypes with human counterparts.

Because human TGF-β3 and murine TGF-β3 are identical in amino acid sequence, individual affinity measurements for these two proteins were not calculated. Similarly, mouse and rat TGF-β1 and 2 are identical in amino acid sequence, and individual affinity measurements were not calculated.

The k_{_a} , k _{_d} , and K_{_D} values of Ab₁, determined by the above methods, are listed in Table 2 below. The K_{_D} values of Ab₁ for human TGF-β1, 2, and 3 were measured to be 1.48, 3.00, and 1.65 nM, respectively. The K_D values of Ab₁ for mouse/rat TGF-β1 and 2 were measured to be 2.80 and 1.88 nM, respectively. These binding properties are similar to those of fresolimumab.

Table 2. Equilibrium constants and affinities of Ab1 for TGF-β

The data above indicate that Ab1 is a potent and selective pan-TGF-β inhibitor. Measurements using surface plasmon resonance showed that Ab1 has an affinity of 1 to 5 nM for all human and murine TGF-β isoforms. High levels of specificity were confirmed by GLP immunohistochemical (IHC) tissue cross-reactivity studies using normal rat, cynomolgus monkey, and human tissues.

Example 2: TGF-β neutralizing efficacy of Ab1

The in vitro potency of Ab1 in neutralizing TGF-β activity was measured in a cell-based assay. This assay measured the ability of TGF-β to inhibit the proliferation of untransformed mink lung epithelial cells (Mv 1Lu cells). See, for example, WO 2006/086469 and Mazzieri et al., eds., “Methods in Molecular Biology,” Vol. 142, “Transforming Growth Factor-β Protocols.” The neutralizing abilities of Ab1, fresolimumab, and 1D11 (a murine anti-TGF-β antibody whose heavy and light chain sequences are disclosed herein as SEQ ID NO: 9 and 10) in human TGF-β1, 2, and 3, and murine TGF-β1 and 2 were evaluated. Recombinant TGF-β proteins were produced internally (human TGF-β1, 2, and 3) or obtained from R&D Systems (murine TGF-β1 and 2).

All human and mouse TGF-β isoforms inhibited mink lung cell proliferation in a dose-dependent manner ranging from 0.02 pg/ml to 10 ng/ml. To quantify the potency of Ab1, fresolimumab, and 1D11, mink lung cells were incubated with 1 ng/ml of the specified TGF-β and serially diluted antibodies. After three days of incubation, cell proliferation was quantified by CyQUANT dye, which fluoresces upon binding to DNA (Figure 1A-E). The data showed that Ab1, fresolimumab, and their mouse alternative 1D11 inhibited all human and mouse TGF-β isoforms to similar degrees.

Example 3: Inhibition of induced T regulatory cell differentiation by Ab1

Regulatory T cells (Tregs) are immunosuppressive and have been associated with negative outcomes in cancer patients. In the following study, we investigated whether Ab1 could inhibit TGF-β-induced differentiation of human CD4 ⁺ T cells into inducible regulatory T cells (iTregs). Primary human CD4 ⁺ T cells were isolated from healthy normal donors. Human TGF-β1 was purchased from R&D Systems.

To investigate the antagonistic activity of Ab1 against endogenously produced TGF-β from cultured cells, total CD4+ T cells were treated for 6 days without the addition of exogenous TGF-β using 50 μg/ml of isotype control (human IgG4, κ anti-chicken egg lysozyme (HEL) antibody, Crown Biosience), Ab1, or fresolimumab, with or without stimulation (anti-CD3, anti ^- CD28, and IL-2), followed by flow cytometry analysis. The mean percentage and standard deviation of the CD25 ⁺ FOXP3 ⁺ population were calculated from triplicate of the parental population (lymphocytes/live/single cells/CD4 ^{+ CD127-} ). Stimulation of total human CD4 ⁺ T cells with anti-CD3, anti-CD28, and IL-2 increased the percentage of FOXP3 ⁺ CD25 ⁺ (iTreg) in the culture from 0% to 15%. Treatment with 50 μg/ml Ab1 or 50 μg/ml fresolimumab reduced the percentage of iTregs to similar degrees (8% and 7%, respectively; Figure 2). Conversely, treatment with an isotype human _IgG4 ( _hIgG4 ) control had the least effect on iTreg differentiation (20% of iTregs) (Figure 2). CD4 ⁺ T cells isolated from a second healthy normal volunteer produced similar results.

To investigate the antagonistic activity of Ab1 against exogenous TGF-β, total CD4 ⁺ T cells incubated with 2 ng/ml human TGF-β1 were treated for 6 days with allotype controls, Ab1, or fresolimumab at different antibody concentrations, in the presence or absence of stimulation (anti-CD3, anti-CD28, and IL-2), followed by flow cytometry analysis. Unless otherwise specified, the mean percentage and standard deviation of the CD25 ⁺ FOXP3 ⁺ population were calculated from triplicate of the parental population (lymphocytes/live/single cells/CD4 ^{+ CD127-} ). Adding exogenous TGF-β1 (2 ng/ml) to stimulated total human CD4 ⁺ T cells increased the percentage of iTregs in the culture from 15% to 55%. Treatment with progressively increasing concentrations of Ab1 concomitantly reduced the percentage of iTregs from 55% to 15% at 200 μg/ml and 43% at 6.25 μg/ml. Treatment with fresolimumab reduced the percentage of iTregs to a similar extent to Ab1 (from 55% to 16% at 200 μg/ml and from 55% to 32% at 6.25 μg/ml). Treatment with different concentrations of the isotype control antibody had no effect on the percentage of iTregs, remaining at 60% at both 200 μg/ml and 6.25 μg/ml. See Figure 3. Similar results were obtained from CD4 ⁺ T cells isolated from a second healthy, normal volunteer.

This study demonstrates that Ab1 inhibits TGF-β-induced iTreg differentiation and may therefore provide clinical benefits by mitigating the immunosuppressive tumor microenvironment.

Example 4: The in vitro effects of the combination of Ab1 and anti-PD-1 antibody

In this study, we investigated whether TGF-β prevented maximal stimulation of T cells in vitro following anti-PD-1 treatment, and if so, whether Ab1 could counteract this prevention. The level of T cell activation was measured using luciferase expression in an expression construct under transcriptional control of a regulatory sequence from NFATc (nuclear factor, cytoplasmic 1, activating T cells).

We used a cell assay system purchased from Promega for this study. This system contained two cell types: 1) Jurkat T cells expressing human PD-1 and a luciferase reporter gene driven by NFAT response elements, and 2) CHO-K1 cells expressing human PD-L1 and engineered cell surface proteins designed to activate homologous T cell receptors in an antigen-independent manner. In co-culture, Jurkat T cells interacted with CHO-K1 cells, resulting in T cell receptor stimulation and NFATC translocation to the nucleus, where it drives luciferase expression. However, the involvement of PD-1/PD-L1 recruited the non-receptor tyrosine protein phosphatase 11 (SHP2) to the T cell receptor complex, inhibiting NFATC nuclear translocation and subsequent luciferase expression. Blockade of PD-1 signaling alleviated SHP2-dependent inhibition and thus allowed maximal luciferase expression. Therefore, this system provides a functional approach for determining the role of TGF-β in T cell signaling and the effect of Ab1 on anti-PD-1 therapy of T cells.

Due to the slow kinetics associated with TGF-β-dependent effects, Jurkat T cells were pretreated with TGF-β prior to T cell receptor stimulation. Human TGF-β1 was purchased from R&D Systems. The isotype control antibody for Ab1 (anti-HEL hIgG ₄ ) was purchased from Crown Bioscience (catalog number C0004-5). Mouse anti-hPD-1 IgG and its isotype control antibody were purchased from BioLegend (catalog number 329912). Fourteen replicates were analyzed for each sample.

The results showed that adding anti-hPD-1 antibody to Jurkat T cells co-cultured with CHO-K1 cells for 24 hours induced luciferase activity (865,794 relative luminescent units [RLU]) to a greater extent than adding the isotype control (234,963 RLU, fold change = 3.685, p < 0.0001) or simply not having the antibody (206,043 RLU, fold change = 4.202, p < 0.0001). Pretreatment of Jurkat T cells with 18 ng/ml TGF-β1 for 12 days in CHO-K1 cell co-cultures containing anti-hPD-1 antibody induced less luciferase activity (638,866 RLU) compared to Jurkat T cells not treated with TGF-β1 (865,794 RLU, fold change = -1.355, p < 0.0001) (Figure 4).

To assess the antagonistic efficacy of Ab1, Jurkat T cells were pretreated with 18 ng/ml TGF-β1 for 12 days in the presence of Ab1, allotype control Ab, or without Ab, and then co-cultured with CHO-K1 cells for 24 hours in the presence of anti-PD-1. Compared with the allotype control Ab (639,440 RLU, fold change = 1.445, p < 0.0001) and the no-Ab control (638,866 RLU, fold change = 1.447, p < 0.0001), the presence of Ab1 (924,186 RLU) moderated the TGF-β-dependent inhibition of luciferase activity. Control groups treated with Ab1 (975654 RLU) or the isotype control (955717 RLU) were added to Jurkat T cells that were not pretreated with TGF-β1 and co-cultured with CHO-K1 cells in the presence of anti-PD-1Ab. Compared with the control without Ab, the control group showed a statistically significant increase in luciferase activity, but with the smallest fold change (865794 RLU, fold change = 1.127 and 1.104, p = 0.0023 and 0.001284, respectively) (Figure 4). RLU values are also listed in Table 3 below.

Table 3. Relative luminescent units in T cell activation assay

To rule out the possibility that TGF-β1 pretreatment of Jurkat T cells might have resulted in reduced proliferation or viability, and thus reduced luciferase activity during 24 hours of co-culture with CHO-K1 cells, we incubated Jurkat T cells with 18 ng/ml TGF-β1 or PBS in the presence of Ab1, anti-HEL hIgG ₄ , or no antibody (vector) for 7 days. Every 2 to 3 days, fresh culture flasks were seeded with equal volumes of each group, at which point both TGF-β1 and antibody were refreshed (a total of two reseeding events occurred). Evaluation of the final cultures showed that viability remained unchanged across all treatment groups (ranging from 94% to 96%). Furthermore, the total number of Jurkat T cells in the final cultures of each treatment group was very similar (ranging from 25 million to 29 million).

The above studies indicate that the increase in downstream signaling of T cell receptors following anti-PD-1 therapy is inhibited by TGF-β, leading to sub-optimal T cell stimulation. Our data suggest that inhibiting TGF-β alleviates the immunosuppressive tumor microenvironment and allows checkpoint modulators such as anti-PD-1 agents to better induce immune responses, thereby increasing the proportion of patients who benefit from immuno-oncology therapy.

Example 5: The effect of the combination of Ab1 and anti-PD1 antibodies in vivo

Next, we investigated the effects of combined anti-TGF-β and anti-PD-1 therapy in a C57BL/6 mouse cancer model.

Tolerability/Initial Safety

Materials and methods

The tolerability of Ab1 and anti-mouse PD-1 (mPD-1) monoclonal antibodies (mAbs) as single agents and in combination was evaluated in female C57BL/6 mice. Ab1 (10, 20, and 50 mg/kg) or an isotype control Ab (anti-HEL hIgG ₄ , purchased from Crown Bioscience; used at 10 and 20 mg/kg) was administered IV every three days (Q3D) as a single agent or in combination with anti-PD1 Mab at 5 mg/kg IV twice weekly. The anti-PD1 Ab used in this study was named “antimPD1_hyb_RMP114_mIgG1LCfullrat” (or x anti-mPD-1Mab). It is a chimeric rat anti-mPD-1 antibody generated by replacing the rat Fc region _{of rat IgG 2a clone} RMP1-14 (BioXcell, catalog number BE0146) with the rat Fc region of mouse IgG 1. The heavy and light chain amino acid sequences of this chimeric antibody are shown in SEQ ID NO:7 and 8. Tolerance was assessed by measuring animal weight and clinical observations. At the end of the three-week treatment period, four hours after the last treatment, terminal sampling was performed, and tissues (heart, kidneys, liver, lungs, and spleen) were fixed in formaldehyde and sent for histopathological analysis.

A dose is considered excessively toxic if it produces 15% weight loss over a three-day period, 20% weight loss over a one-day period, or 10% or more drug-related deaths in a single mouse, unless tumor-induced cachexia leading to weight loss is observed in the control vector-treated group. Animal weight includes tumor weight.

Toxicity/Safety Results

Tolerance studies in C57BL/6 mice showed that single-drug administration at all tested dose levels, as well as the combination of Ab1 and x-anti-mPD-1Mab, was well tolerated. No significant changes in body weight were observed in any treatment group at any tested dose. No serious or major clinical outcomes were observed during the study. Histopathological analysis identified an increase in lymphocyte count in the spleen (white pulp) of all treatment groups, including those treated with the isotype control antibody, regardless of the combination and without any dose relationship. No other significant microscopic findings were observed. Two mice from the combination group (isotype control Ab (10 mg/kg) and anti-PD-1Mab (5 mg/kg)) were found dead after the final administration on the last day of the study. Histopathological analysis did not identify any drug-related cause of death.

Efficacy study

The efficacy of combination therapy with anti-TGF-β and anti-PD-1 was evaluated in C57BL/6 mice carrying subcutaneous MC38 syngeneic colon tumors. Mice were administered 25 mg/kg of Ab1, 5 mg/kg of x-anti-mPD-1Mab, or both, Q3D for three weeks. This study demonstrated that the combination of Ab1 and anti-mPD-1Mab exhibited significantly higher antitumor activity than either agent alone. The materials, methods, and data from this study are described in detail below.

Materials and methods

animal

Female C57BL/6 mice were obtained from Charles River Labs (Wilmington, MA, USA). Animals were acclimatized for at least 3 days prior to study registration. At the start of the study, the mice were 11 weeks old and weighed between 17.0 and 20.9 g. They had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water, and were housed on a 12-hour light/dark cycle.

Tumor cells

MC38 is a colon adenocarcinoma cell line. Cells were obtained from the National Cancer Institute (Bethesda, MD, USA) and cultured in complete medium (CM) at 37°C and 5% _CO2 , consisting of Roswell Park Memorial Institute Medium (RPMI)-1640 with L-glutamine (Gibco, catalog 11875) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) (Gibco, catalog 10438026). Cells were harvested and resuspended in Dulbecco phosphate-buffered saline (DPBS) (Gibco, catalog 14190), and 1 x ^10⁶ cells/200 μl were subcutaneously (SC) implanted into the right side of female C57BL/6 mice.

compound

Ab1 was administered to animals in aqueous solution. It was filtered through PES at 0.22 μm and stored as sterile aliquots at 2–10 °C. The antibody was administered to animals at 10 ml/kg intraperitoneally (IP) or 25 mg/kg.

Anti-HEL hIgG ₄ (Crown Bioscience) was used as an isotype control for Ab1. This antibody was administered to control animals at a dose of 10 ml/kg via intraperitoneal injection (IP), and 25 mg/kg was administered at the same dose.

x-anti-mPD-1Mab (as above) was provided in DPBS (Gibco, catalog number 14190-094) and administered to animals via IP at 10 ml/kg and 5 mg/kg.

Research Design

On day 0, MC38 tumor cells were implanted into 60 animals. On day 8 post-implantation, mice with an average tumor size of 50-75 ^mm³ were pooled and randomly assigned to the control and treatment groups (n=10 per group). Treatment with the aforementioned doses of the vector (PBS, pH 7.2), anti-HEL hIgG ₄ , Ab1, and anti-mPD-1Mab was initiated on day 9 and repeated on days 12, 15, 18, 21, and 27. Animals treated with the vector and anti-HEL hIgG ₄ served as controls. Mice were examined daily, and adverse clinical reactions were recorded. Each mouse was weighed three to four times per week until the end of the experiment.

Mice were euthanized when pathological condition or weight loss ≥20% was observed. Tumors were measured twice weekly with calipers until final sacrifice. Animals were euthanized and the date of death was recorded when the tumor size reached approximately 2000 ^mm³ or when health problems were present (ulceration of 20% of the tumor area). The volume of the solid tumor was estimated from the two-dimensional tumor measurements and calculated according to the following equation:

Tumor volume ( ^mm³ ) = [length (mm) x ^width² ( ^mm² )] / 2

The median percentage regression for a given day is then obtained by taking the median individual percentage regression calculated for each animal in that group on that day. The calculation date is determined on the day on which ΔT/ΔC (i.e., the median change in tumor volume from baseline between the treatment and control groups) is calculated, unless median percentage regression does not represent activity in that group. In that case, the day on which the median percentage regression is greatest is determined. Regression is defined as partial (PR) if the tumor volume decreases to 50% of the tumor volume at the start of treatment. Complete regression (CR) is considered to have been achieved when the tumor volume is below 14 ^mm³ or cannot be recorded.

effect

The primary efficacy endpoint was the change in tumor volume from baseline, indicated by ΔT/ΔC, median percentage regression, partial regression, and complete regression. Tumor volume change was calculated daily for each animal in both the treatment group (T) and the control group (C) by subtracting the tumor volume on the day of the first treatment (staging day) from the tumor volume on the designated observation day. The median ΔT for the treatment group and the median ΔC for the control group were calculated. The ratio ΔT/ΔC was calculated and expressed as a percentage.

ΔT/ΔC = (median ΔT/median ΔC) x 100

A ΔT/ΔC ratio ≤ 40% is considered therapeutically active. A ΔT/ΔC ratio of 0% is considered tumor arrest. A ΔT/ΔC ratio < 0% is considered tumor regression.

The percentage of tumor regression was defined as the percentage reduction in tumor volume in the treatment group on a specified observation day, compared to the tumor volume at the start of the study (t _₀ ). At a specific time point (t) and for each animal, the percentage regression was calculated using the following formula:

% decay (at time t) = [(volume _t0 – volume _t ) / volume _t0 ] x 100

The median percentage regression for a given group is then calculated by taking the median of the individual % regression values calculated for each animal in the group. The date of calculation is determined by the date on which ΔT/ΔC was calculated, unless the median percentage regression does not represent the activity of the group. In that case, the day is determined by the first day when the median percentage regression is at its maximum.

Statistical analysis

A two-way ANOVA with factor treatment and day (repeated) was performed to assess changes in tumor volume compared to baseline. In cases of significant treatment-day interaction or treatment effect, a Bonferroni-Holm corrected multiplicity analysis was subsequently performed to compare all treatment groups with controls on each day from day 8 to day 27. Changes in tumor volume relative to baseline were calculated for each animal and on each day by subtracting the tumor volume on the first treatment day (day 8) from the tumor volume on the specified observation day.

Because heterogeneity of differences was observed between groups, a composite symmetric (CS) covariance structure with the group = option was selected for the ANOVA-type model (SAS Institute Inc. (2008) SAS/STAT 9.2 User Guide, Cary NC). In Figures 5 and 6, the median and median absolute deviation (MAD) for each group are presented for each measurement day. The median and normalized MAD (nMAD = 1.4826 * MAD) for each group are reported in Tables 4-6 below for each measurement day. All statistical analyses were performed using SAS version v9.2 software. A probability less than 5% (p < 0.05) was considered significant.

Efficacy Results

Treatment of tumor-bearing C57BL/6 mice with Ab1, anti-PD-1Mab, or a combination of both was well tolerated and non-toxic, as evidenced by the general health and activity of the animals and the absence of significant changes in body weight. As single agents, 25 mg/kg Q3D of Ab1 and 5 mg/kg Q3D of anti-PD-1Mab resulted in minimum weight loss values of only 3.4% (day 9) and 2.1% (day 9), respectively. The combination of Ab1 (25 mg/kg Q3D) and anti-PD-1Mab (5 mg/kg Q3D) was also well tolerated, showing a minimum weight loss value of 1.3% (day 9) (Table 4).

As single agents, Ab1 (25 mg/kg Q3D) and anti-PD-1Mab (5 mg/kg Q3D) showed no disturbance to tumor growth compared to animals treated with the Ab1 isotype control (anti-HEL hIgG ₄ ). The ΔT/ΔC ratios on day 27 of treatment were 93% and 109%, respectively (Table 4). The combination of anti-PD-1Mab and anti-HEL hIgG ₄ showed minimal antitumor activity, with a ΔT/ΔC of 31% on day 27 of treatment (not statistically different from the control group), and complete regression was observed in only 2 out of 10 mice. However, the combination of anti-PD-1Mab and Ab1 demonstrated superior antitumor activity from day 15 to day 27, with a -1 ΔT/ΔC on day 27 of treatment (statistically different from the control group), and complete regression was observed in 6 out of 10 mice (Table 4).

Table 4. Activities of Abl and X anti-mPD-1Mab in the C57BL/6MC38 cancer model

*ΔW represents the average weight change for each group at the lowest point.

**After repeated measures of two-dimensional ANOVA-Type analysis of tumor volume changes from baseline, multiplicity was adjusted using Bonferroni-Holm, and p-values were obtained using comparative analyses comparing each treatment group with the control group. A probability less than 5% (p<0.05) was considered significant.**

Tables 5 and 6, as well as Figures 5-7, provide additional data showing the activity of antibodies, alone or in combination, against tumor volume in mouse models.

Table 5 Comparison between the treatment group and the control group

*Multiplicity was adjusted using Bonferroni-Holm after two-way Anova-Type analysis of tumor volume changes from baseline, and p-values were obtained using daily comparative analysis relative to controls.

Table 6 Comparison of Ab1 and X-anti-mPD-1Mab as single agents and in combination

*After performing bidirectional Anova-Type analysis on changes in tumor volume from baseline, multiplicity was adjusted using Bonferroni-Holm, and p-values were obtained by comparing the daily combination of Ab1, anti-HEL hIgG ₄ , and x-anti-mPD-1 with the doses of each individual agent involved in the combination.

The data in the tables and figures show that the combination of Ab1 at 25 mg/kg Q3D and x-anti-mPD-1Mab at 5 mg/kg Q3D had a higher antitumor effect than either antibody at those doses. When the combination was compared with Ab1 as a single agent, the p-values at days 19, 23, and 27 were 0.0007, 0.0007, 0.0008, 0.0009, and 0.0008, respectively.

The differences were statistically significant (p < 0.0001 and < 0.0001). When comparing the combination to x-anti-mPD-1Mab as a single agent, the p values at days 19, 23, and 27 were 0.0276, 0.0004, and 0.0024, respectively (Table 6). For the combination of 25 mg/kg Q3D anti-HEL hIgG4 and 5 mg/kg Q3D x-anti-mPD-1Mab, there was no significant difference in treatment efficacy regarding changes in tumor volume from baseline on any day compared to either agent alone.

In summary, from day 15 to day 27, the combination of Ab1 at 25 mg/kg Q3D and anti-mPD-1Mab at 5 mg/kg Q3D had a significantly greater antitumor effect than either drug alone.

In another study, we evaluated the antitumor activity of Ab1 at doses of 1, 10, or 25 mg/kg versus a mouse PD-1 antibody at dose of 5 mg/kg against a subcutaneous MC38 colon cancer model in C57BL/6J mice. Exponentially growing MC38 colon adenocarcinoma cells (NCI, Frederick, MD) were cultured in RPMI-1640 supplemented with 10% FBS in a humidified 5% CO2 incubator and then subcutaneously implanted (1 x 10⁶ cells) into the flank of female C57/Bl6J mice (Jackson Laboratory, BarHarbor, ME). Once the tumors reached an average size of 50–75 mm³, the mice were pooled and randomly assigned to control and treatment groups (n=10 per group). Mice with tumors were then treated intraperitoneally with PBS, an IgG4 isotype control antibody (25 mg/kg), or Ab1 (1, 10, and 25 mg/kg) three times weekly until each animal received a total of 6 to 7 doses. The tumor was measured twice a week using digital calipers, and the tumor volume (mm3 = L x W x H) was calculated and plotted using GraphPad Prism. Mice were euthanized with CO2 at the end of the study if the tumor grew to >2000 mm3 or if the tumor showed ulceration of >20% of the tumor area.

As single agents, Ab1 at a dose of 25 mg/kg Q3D and mouse α-PD-1 antibody at a dose of 5 mg/kg showed partial activity with complete regression in mice carrying MC38 tumors, at 2/8 and 4/8 doses, respectively. Combinations of Ab1 at 1, 10, or 25 mg/kg Q3D with mouse α-PD-1 antibody at 5 mg/kg Q3D showed therapeutic activity. At day 24 post-implantation, when comparing tumor volume change relative to baseline, the combination of all tested doses of Ab1 and mouse α-PD-1 antibody at 5 mg/kg Q3D was more effective than any single agent, with complete regression in 5/8, 6/8, and 7/8 doses for Ab1 at 1, 10, and 25 mg/kg, respectively. Table 6A provides a summary of the results.

Table 6A: Antitumor effects of the Ab1 + anti-mPD-1 mAb combination

In summary, these preclinical data suggest that the combination of PD-1 inhibition and TGF-β inhibition can suppress tumor growth to a greater extent than checkpoint inhibitors alone.

Example 6: Intratumoral TGF-β1 Levels

Intratumoral TGF-β1 levels were investigated in a BALB/c mouse model of colorectal cancer with subcutaneous xenograft transplantation at LoVo. Starting when the tumor volume was less than 100 ^mm³ , mice were intravenously injected with 10, 25, or 50 mg/kg of Ab1 or the isotype control Mab every 3 days for a total of 8 IV administrations.

Tumor samples stored at -80°C in 2ml plastic tubes with 2.8mm ceramic balls (MoBio 13114-50) were thawed at room temperature. 1ml of cold Meso Scale Diagnostic (MSD) Tris Lysis buffer (R60TX-2) (supplemented with 1x Halt ^™ protease and phosphatase inhibitor mixture (Thermo 78440)) was added to the tissue, and homogenization was performed for 2 cycles at 6500rpm at 4°C for 20s each using a 24Dual homogenizer (Bertin Instruments). The lysate was clarified by centrifugation at 20,000xg for 10 minutes at 4°C in an Eppendorf 5417C centrifuge. As described above, the supernatant was transferred to clean, frozen Eppendorf tubes and further clarified by centrifugation for another 20 minutes. The supernatant was then transferred to plastic 96-well storage blocks, rapidly frozen in liquid nitrogen, and stored at -80°C.

The next day, the samples were thawed at room temperature and placed on ice. Protein concentration in the lysates was measured using the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo 23225) according to the manufacturer's instructions. The lysates were normalized to a protein concentration of approximately 8 mg/mL using MSD Tris Lysis buffer (see above) with protease and phosphatase inhibitors, and aliquoted into plastic microtubes.

The concentration of TGF-β1 in normalized tumor lysates was measured using an electrochemiluminescence assay with a human TGF-β1 kit (MSD, K151IUC-2) according to the manufacturer's instructions. Recombinant mouse TGF-β1 (R&D Systems, catalog number 7666-MB-005), serially diluted in MSD Lysis Buffer, was used as a calibrator. Samples were loaded in duplicate onto plates. Electrochemiluminescence signals were measured using a MESO SECTOR S 600 plate reader (MSD), and the concentration of TGF-β1 in the samples was quantified using MSDDiscovery Workbench software v4.0 based on a standard curve.

The average concentration of the two replicates of the sample was calculated using software. Concentration values identified by the software as "Below Fit Curve Range" or "Below Detection Range" were replaced with zero. To calculate the TGF-β1 concentration per mg of total protein, the concentration measured in the assay (pg/ml) was divided by the protein concentration in the sample (mg/ml).

The results showed that the median level of intratumoral TGF-β1 in mice injected with the isotype control was 21.4 pg/mg of total protein, while the corresponding level in mice injected with Ab1 was undetectable (Figure 8).

To demonstrate the relevance of these findings in humans, we tested intratumoral TGF-β1 levels in 10 human colorectal tumor samples and 10 human melanoma tumor samples using the methods described above. For human CRC samples, TGF-β1 levels ranged from approximately 7 to 25 pg/mg. For human melanoma samples, TGF-β1 levels ranged from approximately 1 pg/mg to as high as 43 pg/mg. These data further support the potential use of anti-TGF-β1 therapeutics such as Ab1, alone or in combination with other immune checkpoint inhibitors such as anti-PD-1 antibodies, in the treatment of tumors.

Example 7: Pharmacokinetic Study of Ab1

This embodiment describes a study characterizing the pharmacokinetic (PK) profile of Ab1 and comparing it with that of fresolimumab. In one study, five groups of jejunal Sprague-Dawley rats were administered a single intravenous dose of 5 mg/kg of either Ab1 or fresolimumab. Each group consisted of five females and five males. Blood samples were collected from the rats at 0.25, 6, 24, 48, 72, 144, 192, and 240 hours post-administration. Serum concentrations of Ab1 and fresolimumab were determined by ELISA. Comparability was established if the 90% confidence interval for the AUC ratio (of the test substance versus the reference substance) was within the range of 80% to 125%.

Figure 9A shows the changes in antibody serum concentrations over time from five groups of rats. PK parameters from groups 2, 4, and 5 (see the legend for Figure 9A) are shown in Table 7 below. This study showed that Ab1 exhibited linear PK performance, with a much longer half-life (mean T _1/2 of 7.1 days vs. 4.3 days) and a lower elimination rate (CL of 0.30 ml/hr/kg vs. 0.51 ml/hr/kg) than fresolimumab. The data showed that Ab1 provided 1.7 times higher exposure to fresolimumab in rats.

Table 7. PK comparison between Fresolimumab and Ab1

The mean ± SD of the group was statistically different from that of the “fresolimumab(B2)” group (group 2 in Figure 9A).

Another pharmacokinetic (PK) study on Ab1 was conducted in cynomolgus monkeys (Study 2). Five females and five males were in each group, and administered a single intravenous infusion of Ab1 at 1 mg/kg (Fig. 9B) or 10 mg/kg (Fig. 9D), or five weekly doses of Ab1 at 1 mg/kg (Fig. 9C) or 10 mg/kg (Fig. 9E). Figures 9B-E show changes in serum Ab1 concentrations over time in the monkeys. Serum concentrations of fresolimumab administered to monkeys in a previous study at a single or repeated Q2W (bi-weekly) dose are also shown in the figures for comparison. These data indicate that Ab1 also exhibits linear pharmacokinetic behavior in monkeys after single and repeated administrations, and shows higher exposure than fresolimumab at both 1 mg/kg and 10 mg/kg doses. At a single dose of 10 mg/kg, Ab1 has a half-life of 13 days, while fresolimumab has a half-life of 4.5 days; Ab1 has a CL of approximately 0.40 ml/hr/kg, while fresolimumab has a CL of 0.66 ml/hr/kg. Similar to rat studies, monkey studies also showed that Ab1 provides approximately 1.7 times higher exposure than fresolimumab.

The above studies show that Ab1 has a statistically significant longer half-life, longer clearance time, and higher biological exposure in vivo compared to fresolimumab.

Furthermore, studies in Balb/C mice carrying Ab12 tumors showed that Ab1 had a similar PK profile, regardless of whether it was administered intravenously or intraperitoneally.

Using allometric scaling on a two-compartment model, we predicted the following PK parameters in 70kg males based on monkey data (Table 8):

Table 8 Alloch modeling of PK parameters

PK parameters Ab1 in monkeys Ab1 in the human body <![CDATA[T_1/2(days)]]> 13.1 20.9 CL (ml/hr/kg) 0.392 5.7 V1 (Central Room) (L) 0.104 2.43 Q(ml/hr) 3.18 46 V2 (Surrounding Room) (L) 0.0693 1.62

In humans, the predicted PK parameters of Ab1 are also more favorable than those of fresolimumab. For example, in humans, the clearance rate of fresolimumab is 12.3 ml/hr/kg, which is faster than that of Ab1.

Example 8: Toxicological study of Ab1

Toxicological studies of Ab1 were conducted in rats and cynomolgus monkeys. Safety pharmacological endpoints were assessed weekly for up to 5 weeks in a repeated-dose, GLP (Good Laboratory Practice) setting. No Ab1-related histopathological findings were observed at the injection site at doses up to 10 mg/kg/dose (2 mg/ml) in monkeys and up to 30 mg/kg/dose (6 mg/ml) in rats. No Ab1-related effects were noted at any dose level tested for neurological examination, body temperature, respiratory rate, blood pressure, and ECG parameters in this study.

The NOAEL (No Observed Adverse Effect Level) in rats was found to be 3 mg/kg/dose with repeated weekly administration for 5 weeks, and the STD10 (Severe Toxic Dose) in rats was found to be 3 to 10 mg/kg/dose. Toxicity included valvular hyperplasia characterized by multiple thickened nodules; and abnormal pulmonary conditions such as mixed-cell alveolar exudate, mixed-cell perivascular infiltration, muscular arterial hypertrophy, hemorrhage, and/or increased lung weight.

The NOAEL and HNSTD (i.e., the highest non-serious toxic dose above which lethal, life-threatening, or irreversible toxicity occurs) in monkeys administered the drug weekly for 5 weeks were found to be 10 mg/kg/dose (refer to fresolimumab, whose NOAEL in monkeys was 1 mg/kg when administered 7 or 13 times every two weeks or Q3D for 4 weeks). See also the data in Table 9 below.

Table 9 Summary of toxicological studies in rats and monkeys

Based on the above toxicological data, it is expected that Ab1 can be safely administered to human patients at a dose level of approximately 0.05 mg/kg to 0.5 mg/kg weekly or at a lower frequency, such as every two weeks.

Example 9: In vivo efficacy of anti-TGF-β monotherapy

In this study, we investigated the effects of 1D11 (a mouse _IgG1 anti-bovine TGF-β antibody that cross-reacts with human and mouse TGF-β1, 2, and 3) on a metastatic syngeneic tumor model. In this model, B16-F10 mouse melanoma cells were introduced intravenously into the footpads of C57BL/6 mice, resulting in metastasis in the draining lymph nodes. Although treatment with the control antibody 13C4 was ineffective, treatment with 50 mg/kg 1D11 three times weekly, starting one day after tumor inoculation, completely eliminated the metastasis.

To investigate the role of the immune response, B16-F10 was implanted into the footpads of mice with a defective β2-microglobulin gene, thus lacking a CD8 ⁺ cytotoxic T-cell response, and pretreated as before. Contrary to results observed in immunocompetent mice, 1D11 had no effect on the number of metastases in the draining lymph nodes of these mice. These results suggest that the mechanism of TGF-β inhibition depends on adaptive cellular immunity.

Example 10: TGF-β Signatures in Cancer

Previous studies have shown that melanoma patients who do not respond to anti-PD-1 therapy possess the transcriptional signature marker IPRES (Hugo et al., Cell (2016) 165:35-44). To investigate the mechanisms of innate resistance to anti-PD-1 monotherapy, we examined the transcriptional signature markers of non-responders relative to responders. We found that comparing these profiles with a database containing over 1 million profiles using GeneSet Enrichment Analyses revealed a strong correlation between anti-PD-1 response and activation of TGF-β signaling in the tumor. These data suggest that TGF-β is associated with innate resistance to anti-PD-1 monotherapy at baseline in melanoma.

Furthermore, we found a strong correlation between anti-PD-1 response and TGF-β signaling activation (R = 0.59, p-value < 9E-4 for t-test). Therefore, we arrive at our gateway indication: TGF-β-mediated immunosuppression in melanoma (e.g., metastatic melanoma) may lead to innate resistance. Additionally, we found that TGF-β-induced gene expression changes could be quenched by 1D11 treatment, confirming the specificity of the TGF-β activation signature marker. These results support the use of combination anti-TGF-β and anti-PD-1 therapies for cancer patients unresponsive to anti-PD-1 monotherapy.

Analysis of this correlation across tumor types other than melanoma revealed that mesenchymal tumors (e.g., CRC, HCC, head and neck squamous cell carcinoma, and ovarian cancer) are also rich in both TGF-β activation and predicted anti-PD-1 resistance. This finding is consistent with the role of TGF-β signaling in EMT. Therefore, we arrive at our second threshold: mesenchymal tumors, particularly those with immune infiltration, may benefit from combination therapy with anti-TGF-β and anti-PD-1. Using machine learning methods, a smaller number of genes, such as ACTA2, VIM, MGP, ZEB2, and ZWINT, were identified from over 30 EMT biomarker genes that could be used for selecting mesenchymal tumors. For example, ACTA2 and VIM were found to be translocate across tumor types. Therefore, TGF-β activation transcriptional signature markers and genes within these signature markers could serve as useful biomarkers for selecting cancer patients for combination therapy with anti-TGF-β and anti-PD-1 antibodies at baseline.

To investigate biomarkers in the tumor microenvironment, the immune status of patient tumors was assessed using MultiOmyx, a multiplex IHC assay for CRC and melanoma. Twelve biomarkers (together describing 22 immune cell types) were multiplexed on individual FFPE slices from each tumor sample. The study included a series of inflammation assays to assess how well the analytes evaluated each tumor type and how well they were associated with potential treatment efficacy. Statistical methods were developed to evaluate differences at the cell population level, including consistency of replication, volcano plots of ANOVA, and correlation matrices. The MultiOmyx assay demonstrated superior technical reproducibility and accuracy, favorable dynamic range, differences in inflammatory status across selected immune cells and regions of interest, and included both positive and negative correlations between cell populations.

Example 11: Changes in TGF-β1, MIP2, and KC/GRO in MC38 tumors after Ab1 treatment with or without anti-PD1.

To demonstrate the neutralization of TGF-β, the ability of Ab1 (with or without anti-PD-1) to affect cytokine expression in tumors was evaluated.

When the tumor volume was 61–110 ^mm³ , MC38 tumor-bearing mice were treated with a single dose of PBS or anti-PD-1 alone (5 mg/kg) or an increased dose of Ab1 (10, 25, or 50 mg/kg, intraperitoneally) in combination with anti-PD-1 (5 mg/kg). Tumors were harvested at 1, 6, 10, 24, 72, and 168 hours post-treatment and rapidly frozen in 2 ml plastic tubes (Precyllys KT3961-1007.2) with 2.8 mm ceramic balls and stored at -80°C. Tumors were thawed at room temperature to prepare lysates. Add 1 ml of cold Meso Scale Diagnostics (MSD) Tris lysis buffer (R60TX-2) supplemented with a 1x Halt ^™ protease and phosphatase inhibitor mixture (Thermo 78440) to the tissue, and then homogenize using a 24-Dual homogenizer (Bertin Instruments) at 6500 rpm for 20 s per cycle at 4°C. Clarify the lysate by centrifuging at 20,000 x g for 10 min at 4°C in an Eppendorf 5417C centrifuge. As described above, transfer the supernatant to clean, frozen Eppendorf tubes and further clarify by centrifugation for another 30 min. Then, transfer the supernatant to plastic 96-well storage blocks and prevent on ice. Measure the protein concentration in the lysate using the Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo 23225) according to the manufacturer's instructions. The lysates were normalized to a protein concentration of approximately 5 mg/ml using MSD Tris Lysis buffer (see above) containing protease and phosphatase inhibitors, aliquoted into plastic microtubes, rapidly frozen in liquid nitrogen, and stored at -80°C.

The concentration of active TGF-β1 in tumor lysates was measured using an electrochemiluminescence assay with a human TGF-β1 kit (MSD, K151IUC-2). Standardized tumor lysates prepared as described above were melted and assayed according to the manufacturer's instructions. To represent the quantity of only the active form of TGF-β1 present in the tumor, rather than total TGF-β1 (which includes TGFβ-1 forming complexes with potential related peptides), no acid treatment was performed on the samples. Samples were loaded in duplicate onto plates. Electrochemiluminescence signals were measured using a MESO SECTOR S 600 plate reader (MSD), and the TGF-β1 concentration in the samples was quantified based on a standard curve using MSD Discovery Workbench software v4.0.

Animals treated with Ab1 at all dosing levels (10, 25, or 50 mg/kg) in combination with anti-PD-1 (5 mg/kg) showed reduced levels of active TGF-β1 in tumors compared to animals treated with PBS or anti-PD-1 alone. This indicates that Ab1 binds to its target in vivo (Figure 10A). The reduction in active TGF-β1 levels was observed within 1 hour and persisted for at least 168 hours.

MIP-2 (CXCL2) and KC/GRO (CXCL1) are chemokines involved in the chemotaxis of granulocytes, including neutrophils. The levels of MIP2 and KC/GRO were also assessed in these same samples. In animals treated with Ab1 in combination with anti-PD-1, compared to animals treated with PBS or anti-PD-1 alone, the intratumoral levels of MIP2 increased at least fourfold; and the increase in MIP-2 levels persisted for at least 168 hours (Fig. 10B). Similarly, compared to the levels of MIP-2, the levels of KC/GRO increased, but at later time points of 72 and 168 hours (Fig. 10C). Therefore, the combination of Ab1 and anti-mPD-1 mAb induced a decrease in active TGF-β1 levels earlier than the increases in MIP2 and KC/GRO levels. These results suggest that Ab1 can reduce the levels of TGF-β in the tumor microenvironment and inhibit TGF-β within the tumor microenvironment. Furthermore, the observed increases in MIP2 and KC/GRO levels suggest that these are cytokines affected by the neutralization of TGF-β and could therefore serve as potential biomarkers in patients treated with Ab1.

Example 12: Restoration of NK cell aggregation by Ab1 treatment

TGF-β is known to affect the immune system by inhibiting the activity of different immune cell types. TGF-β has been reported to inhibit natural killer (NK) cell activity and NK cell-mediated ADCC (Trotta et al., Journal of Immunology (2008) 181:3784-3792). Recently, the formation of dense clusters of NK cells has been reported as a mechanism by which IL-2 localizes within these densely packed clusters to enhance NK cell activity and activation (Kim et al., Scientific Reports (2017) 7:40623). Purified human NK cells cultured in vitro in the presence of IL-2 were shown to form these densely packed clusters.

In this study, we evaluated the effect of TGF-β on NK cell aggregation in the absence or presence of Ab1. NK cells were freshly isolated from the blood of healthy donors using the NK cell RosetteSep reagent according to the manufacturer's protocol (Stem Cell Technologies). NK cells were cultured at 1.2 x ^10⁵ cells/well in round-bottom assay plates (Costar) supplemented with Myelocult (Stem Cell Technologies) IL-2 (100 IU/mL). As indicated, TGF-β1 was added to a final concentration of 0.1, 1, or 10 ng/mL in the presence of irrelevant IgG4 or Ab1 (100 μg/mL). Cells were cultured for 72 hours, and NK cell aggregation was visualized by capturing images on a Nikon microscope.

The addition of escalating doses of TGF-β1 inhibited NK cell aggregation. When Ab1, instead of the IgG4 control antibody, was added to NK cell cultures, NK cell cluster formation was observed. These results suggest that TGF-β neutralization affects NK cell activation, leading to increased NK cell activity and proliferation to support the immune system's anti-tumor response.

Example 13: Reversal of Ab1 treatment on TGF-β-mediated inhibition of IFN-γ production in proliferating CD8 ⁺ T cells

Besides the innate immune system, TGF-β has been reported to suppress CD8 ⁺ T cell activity (Flavell et al., Nature Reviews Immunology (2010) 10:554-567). To explore the effects of TGF-β and Ab1 on CD8 ⁺ T cell activity, an MLR (mixed lymphocyte reaction) assay system was established in which purified human CD3 ⁺ cells were mixed with BLCL cells. CD8 ⁺ cell proliferation and IFN-γ production were first assessed in the presence of TGF-β. Specifically, CD3 ⁺ cells were isolated from PBMCs graded from normal healthy donors using the EasySep T Cell Enrichment Kit (StemCell Technologies) after Ficoll gradient separation. The CD3 ⁺ cells were then labeled with CellTrace Violet according to the manufacturer's protocol (ThermoFisher). MLR assays were performed by mixing labeled CD3 ⁺ cells ( ² x 10⁵ cells) with irradiated BLCL cells (Astarte Bio) (2 x ^10⁴ cells; 2 min) in RPMI supplemented with 10% FBS. TGF-β1, IgG4 control antibody, and/or Ab1 were added to the culture as shown, and the culture was incubated at 37°C and 5% _CO₂ for 4 days. Cells were then stimulated for 4 hours in the presence of a PMA cell stimulation mixture (eBioscience) and a protein transporter inhibitor mixture (eBioscience). Viable cells were distinguished by staining with Zombie NIR viability dye (BioLegend) on ice and washing with FAC buffer. Cells were fixed, washed, pelleted, and resuspended in FAC buffer with True-Nuclear buffer (BioLegend). Cells were prepared for flow cytometry using BV650 anti-huCD4, PERCP/Cy5.5 anti-huCD8, FITC anti-huCD3, and PE anti-huIFNγ (BioLegend) staining. Flow cytometry was run on BD Canto, and the results were analyzed in FlowJo software, with gating of live cells, single cells ^, and CD3 ⁺ cells. The percentage of IFNγ ⁺ CD8 ⁺ T cells was quantified by gating CD8 ⁺ cells, which had proliferated based on reduced CellTraceViolet staining and were positive for IFN-γ staining. FMO was run as a control for all antibody staining.

The presence of TGF-β in the MLR assay showed that the percentage of IFN-γ-positive CD8 ⁺ T cells decreased by approximately 4-fold (Fig. 11A). In the absence of TGF-β, the presence of Ab1 or the control Ab had no effect on the development of these IFNγ ⁺ proliferating CD8 ⁺ cells (Fig. 11B). However, the presence of Ab1, but not the control antibody, was able to restore the proliferation of IFNγ ⁺ CD8 ⁺ cells in a dose-dependent manner. These results suggest that TGF-β neutralization can affect the adaptive immune system by blocking the immunosuppressive effect of TGF-β on the proliferation of IFN-γ-expressing effector CD8 ⁺ cells. These IFNγ ⁺ CD8 ⁺ T cells have been suggested to play an important role in antitumor immunity (Ikeda et al., Cytokine Growth Factor Rev (2002) 13:95-109).

Example 14: Response of an allogeneic mouse model to anti-TGF-β therapy

In this study, we investigated which syngeneic mouse models could be used to predict responses to treatment with anti-TGF-β antibodies Ab1 and anti-PD-1. To stratify the mouse models, we assessed CD8 ⁺ T cell infiltration into mouse tumors and TGF-β pathway activation. CD8 ⁺ T cell infiltration was analyzed based on CD8 ⁺ T cell signature markers derived from RNASeq data. Transcriptomic profiles of 17 different syngeneic mouse models with tumor cells from several indications (shown below the x-axis in Figures 12A and 12B) were constructed using whole-transcriptome RNAseq. This "compendium" of syngeneic models was built on the common background strain C57/BL6, with 5 to 7 biological replicates used for each model. Gene expression profiles, expressed as per million transcripts (TPM), were generated after sequencing with Illumina2000 using the STAR aligner and Cufflinks transcript abundance estimators, following standard processing of the raw sequence reads. Finally, the resulting multi-sample data matrix is quantile normalized.

Figure 12A shows the relative abundance of CD8 ⁺ T cells (log2-converted) in the summary. Relative CD8 ⁺ T cell abundance was estimated using the unique biomarker gene CD8B, which has been shown to be a highly specific indicator of CD8T cell presence (Becht et al., Curr Opin Immunol (2016) 39:7-13; and Becht et al., Genome Biol (2016) 17:218). Each block plot summarizes the range of values in the biological replicates. The MC38 model showed approximately 2-fold more CD8 ⁺ T cell infiltration compared to the EMT6 model (left and right boxes, respectively). The A20 and EL4 lymphoma models showed the highest and lowest overall levels of CD8 ⁺ T cell infiltration, respectively, while CD8 ⁺ T cells were negligible in EL4.

The MC38, MC38.ova, CT26, and L1210 mouse cell lines exhibited the highest levels of the CD8 gene signature marker. Additionally, the EMT-6 breast cancer cell line showed near-baseline T-cell infiltration, consistent with recent reports of an immune-excluded phenotype in EMT6 tumors (S. Mariathasan et al., 2017, ESMOD Immuno-Oncology Congress, Geneva, Geneva, Switzerland).

Figure 12B shows TGF-β pathway activation in the overall profile. Transcriptional signature markers of 170 genes demonstrating TGF-β pathway activation, derived from in vitro TGF-β stimulation of MCF7 cells and validated by comparison with several other TGF-β signature markers, were used to assign pathway activation scores for each profile in the profile. Scores were calculated using “Regulated Gene Set Enrichment Analysis” (rGSEA, Theilhaber et al., 2014) and expressed as log2 enrichment of signature marker genes against the gene background. While the MC38 model showed average activation, the EMT6 model showed very high TGF-β pathway activation (left and right boxes, respectively).

Example 15: Effects of the combination of Ab1 and anti-PD1 antibodies on a mouse model of breast cancer

In this study, we investigated the therapeutic effect of Ab1 with and without anti-PD-1. Exponentially growing EMT-6 mammary cells (CRL-2755, ATCC) were cultured in RPMI-1640 supplemented with 10% FBS in a humidified 5% _CO2 incubator and then subcutaneously implanted (0.5 x ^10⁶ cells/mouse) into the flanks of female BALB/c mice (Shanghai Lingchang Biotechnology Co., Ltd., Shanghai, China). Once the tumors reached a mean size of 68–116 ^mm³ , the mice were pooled and randomly assigned to control and treatment groups (n=10 per group). Each tumor-bearing mouse was then treated intraperitoneally three times weekly with PBS and Ab1 (10 and 25 mg/kg) for a total of six doses. Tumors were measured twice weekly using digital calipers, and tumor volume ( ^mm³ = L x W x H) was calculated and plotted using GraphPad Prism. Mice were euthanized with _CO2 at the end of the study if the tumor grew to >3000 ^mm3 or the tumor showed ulceration of >20% of the tumor area.

As single agents, Ab1 at doses of 10 or 25 mg/kg Q3D and mouse α-PD-1 antibody at dose of 5 mg/kg showed partial activity with 1/10, 2/10, and 2/10 complete regression, respectively, in mice carrying EMT-6 tumors. The combination of Ab1 at doses of 10 or 25 mg/kg Q3D with mouse α-PD-1 antibody at dose of 5 mg/kg Q3D was therapeutically active. At day 31 post-implantation, when comparing changes in tumor volume from baseline, the combination of all tested doses of Ab1 with mouse α-PD-1 antibody at dose of 5 mg/kg Q3D was more effective than any single agent, with 7/10 and 4/10 complete regression for 10 and 25 mg/kg Ab1, respectively. Table 10 summarizes the results.

Table 10 Effects of Ab1/anti-mPD-1 combination on EMT-6 mouse model

Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although similar or equivalent methods and materials may also be used in the practice or testing of this invention. The entire contents of all publications and other references mentioned herein are incorporated by reference. In case of conflict, this specification, including the definitions, shall prevail. While numerous references are cited herein, such citations do not constitute an admission that any of these references constitutes part of common general knowledge in the art. Furthermore, unless the context requires otherwise, singular forms shall include plural forms, and plural forms shall include singular forms. Generally, nomenclature and techniques relating to 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 are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly done in the art or as described herein. Throughout the specification and embodiments, the words “have” and “comprise” or variations such as “has”, “having”, “comprises”, or “comprising” will be understood to imply inclusion of the said integer or group of integers, but do not exclude any other integer or group of integers.

sequence list

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 YKTTPPVLDSDGSFFLYSRL 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 (fresolimumab heavy chain, including leader sequence - residues 1-19)

MGWSCIILFL VATATGVHSQ VQLVQSGAEV KKKPGSSVKVS CKASGYTFSS NVISWVRQAPGQGLEWMGGVIPIVDIANYA QRFKGRVTIT ADESTSTTYM ELSSLRSEDT AVYYCASTLG

LVLDAMDYWG QGTLVTVSSA STKGPSVFPL APCSRSTSES TAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTV PSSSLGTKTY TCNVDHKPSN TKVDKRVESK

YGPPCPSCPA PEFLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG

QPREPQVYTL PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK

SEQ ID NO:4 (fresolimumab light chain, including leader sequence - residues 1-19)

MGWSCIILFL VATATGVHSE TVLTQSPGTL SLSPGERATL SCRASQSLGS SYLAWYQQKPGQAPRLLIYGASSRAPGIPD RFSGSGSGTD FTLTISRLEP EDFAVYYCQQ YADSPITFGQ

GTRLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

SEQ ID NO:5 (anti-PD-1Mab heavy chain)

EVQLLESGGV LVQPGGSLRL SCAASGFTFS NFGMTWVRQA PGKGLEWVSG ISGGGRDTYFADSVKGRFTISRDNSKNTLY LQMNSLK GED TAVYYCVKWG NIYFDYWGQG TLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVL QSSGLY SLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV V DVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQPREPQVYTLPP

SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVDKSRWQEGNVF

SCSVMHEALH NHYTQKSLSL SLGK

SEQ ID NO:6 (anti-PD-1Mab light chain)

DIQMTQSPSS LSASVGDSIT ITCRASLSIN TFLNWYQQKP GKAPNLLIYA ASSLHGGVPSRFSGSGSGTD

FTLTIRTLQP EDFATYYCQQ SSNTPFTFGP GTVVDFRRTV AAPSVFIFPP SDEQLKSGTASVVCLLNNFY

PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQGLSSPVTKSFN RGEC

SEQ ID NO:7 (x-anti-mPD-1Mab heavy chain)

EVQLQESGPG LVKPSQSLSL TCSVTGYSIT SSYRWNWIRK FPGNRLEWMG YINSAGISNYNPSLKRRISI

TRDTSKNQFF LQVNSVTTED AATYYCARSD NMGTTPFTYW GQGTLVTVSS AKTTPPSVYPLAPGSAAQTN

SMVTLGCLVK GYFPEPVTVT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV PSSTWPSETVTCNVAHPASS

TKVDKKIVPR DCGCKPCICT VPEVSSVFIF PPKPKDVLTI TLTPKVTCVV VDISKDDPEVQFSWFVDDVE

VHTAQTQPRE EQFNSTFRSV SELPIMHQDW LNGKEFKCRV NSAAFPAPIE KTISKTKGRPKAPQVYTIPP

PKEQMAKDKV SLTCMITDFF PEDITVEWQW NGQPAENYKN TQPIMDTDGS YFVYSKLNVQKSNWEAGNTF

TCSVLHEGLH NHHTEKSLSH SPG

SEQ ID NO:8 (x-anti-mPD-1Mab light chain)

DIVMTQGTLP NPVPSGESVS ITCRSSKSLL YSDGKTYLNW YLQRPGQSPQ LLIYWMSTRASGVSDRFSGS

GSGTDFTLKI SGVEAEDVGI YYCQQGLEFP TFGGGTKLEL KRADAAPTVS IFPPSTEQLATGGASVVCLM

NNFYPRDISV KWKIDGTERR DGVLDSVTDQ DSKDSTYSMS STLSLTKADY ESHNLYTCEVVHKTSSSPVV

KSFNRNEC

SEQ ID NO:9 (1D11 heavy chain)

HVQLQQSGPE LVRPGASVKL SCKASGYIFI TYWMNWVKQR PGQGLEWIGQ IFPASGSTNYNEMFEGKATL

TVDTSSSTAY MQLSSLTSED SAVYYCARGD GNYALDAMDY WGQGTSVTVS

SAKTTPPSVY PLAPGSAAQT NSMVTLGCLV KGYFPEPVTV TWNSGSLSSG VHTFPAVLQS

DLYTLSSSVT VPSSTWPSQT VTCNVAHPAS STKVDKKIVP RDCGCKPCIC TVPEVSSVFI

FPPKPKDVLT ITLTPKVTCV VVDISKDDPE VQFSWFVDDV EVHTAQTKPR EEQFNSTFRS

VSELPIMHQD WLNGKEFKCR VNSAAFPAPI EKTISKTKGR PKAPQVYTIP PPKEQMAKDK

VSLTCMITDF FPEDITVEWQ WNGQPAENYK NTQPIMDTDG SYFVYSKLNV QKSNWEAGNT

FTCSVLHEGL HNHHTEKSLS HSPGK

SEQ ID NO:10 (1D11 light chain)

NIVLTQSPAS LAVSLGQRAT ISCRASESVD SYGNSFMHWY QQKSGQPPKL LIYLASNLES

GVPARFSGSG SRTDFTLTID PVEDDAATY YCQQNNEDPL TFGAGTKLEL KRADAAPTVS

IFPPSSEQLT SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS

STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC

Claims (11)

1. An isolated nucleic acid molecule encoding a monoclonal antibody that specifically binds to human TGF-β1, TGF-β2 and TGF-β3, said antibody comprising heavy chain complementarity-determining regions (CDRs) 1-3 in SEQ ID NO:1 and light chain CDRs 1-3 in SEQ ID NO:2, wherein said antibody contains a human _IgG4 constant region having a proline at position 228 (EU number). 2. The isolated nucleic acid molecule according to claim 1, wherein the antibody comprises a heavy chain variable domain ( _VH ) amino acid sequence corresponding to residues 1-120 of SEQ ID NO:1 and a light chain variable domain ( _VL ) amino acid sequence corresponding to residues 1-108 of SEQ ID NO:2. 3. The isolated nucleic acid molecule according to claim 1, wherein the antibody has one or more of the following properties: a) Inhibit the differentiation of CD4 ⁺ T cells into inducible regulatory T cells (iTregs), b) Increase CD8 ⁺ T cell proliferation c) Increase the aggregation of natural killer (NK) cells. d) Increase the level of MIP2, and e) Increase the level of KC/GRO. 4. A vector comprising the nucleic acid molecule according to claim 1. 5. A mammalian cell comprising the nucleotide sequence of claim 1 encoding the heavy and light chains of the antibody. 6. A method for producing antibodies, comprising: The mammalian cells of claim 5 are cultured under conditions that allow expression of the nucleotide sequence; and The antibody was isolated from the culture. 7. An isolated nucleic acid molecule encoding a monoclonal antibody, wherein the amino acid sequences of the heavy chain and light chain of the monoclonal antibody comprise SEQ ID NO:1 and SEQ ID NO:2, respectively. 8. A vector comprising the nucleic acid molecule according to claim 7. 9. A mammalian cell comprising the nucleotide sequence of claim 7 encoding the heavy and light chains of the antibody. 10. A method for producing antibodies, comprising: The mammalian cells of claim 9 are cultured under conditions that allow expression of the nucleotide sequence; and The antibody was isolated from the culture. 11. A method for producing a pharmaceutical composition, comprising: A specific monoclonal antibody binding to human TGF-β1, TGF-β2, and TGF-β3 is provided, the antibody comprising heavy chain complementarity-determining regions (CDRs) 1-3 of SEQ ID NO:1 and light chain CDRs 1-3 of SEQ ID NO:2, wherein the antibody contains a human _IgG4 constant region having a proline at position 228 (EU number); and The antibody is mixed with a pharmaceutically acceptable carrier.