Monospecific and multispecific anti-TMEFF2 antibodies and uses thereof

Abstract

The disclosure provided herein relates to monospecific and multispecific anti-TMEFF2 antibodies, and methods of making and using the same.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on January 31, 2019, is named JBI5160WOPCT1_SL.txt, and is 144,156 bytes in size. Technical Field

[0003] The disclosure provided herein relates to monospecific and multispecific anti-TMEFF2 antibodies, and methods of making and using the same. Background Art

[0004] Prostate cancer is the second most common cancer in men worldwide and the sixth leading cause of cancer-related death. Globally, there are approximately 1,100,000 new cases and 300,000 deaths each year, accounting for 4% of all cancer deaths. It is estimated that 1 in 6 men will be diagnosed with the disease in his lifetime. Prostate cancer risk is strongly associated with age: approximately three-quarters of cases occur in men over the age of 65, with the highest number of cases occurring in men aged 70-74 years. Based on estimates from autopsy data, approximately half of men in their 50s and 80% of men in their 80s have histological evidence of prostate cancer. In the early stages, the 5-year survival rate is close to 100%. However, when the cancer has metastasized, the 5-year survival rate drops to 28%, and effective treatments for advanced prostate cancer are still needed.

[0005] Androgen deprivation therapy usually causes the disease to remain stable or regress (in 80% of patients).Current treatment for prostate cancer includes surgery, radiotherapy and hormone therapy.Usually, cancer vaccine sipuleucel-T, radiopharmaceutical agents (such as radium chloride 223), adjuvant hormone therapy (such as abiraterone or enzalutamide) and / or chemotherapy (docetaxel and cabazitaxel) are added to hormone therapy in sequence.Although each of these treatments can cause cancer growth to be delayed for several months and alleviate the symptoms caused by the disease, the progress of metastatic prostate cancer eventually develops.Although testosterone levels are reduced by hormone therapy, when prostate cancer also grows, treatment options are limited.

[0006] Therefore, there is a need to develop additional therapeutic agents to treat prostate cancer. Summary of the invention

[0007] The present invention provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane proximal region of SEQ ID NO: 110 of TMEFF2.

[0008] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane proximal region of TMEFF2, wherein the antibody or the antigen-binding fragment thereof competes with a reference antibody for binding to the membrane proximal region of TMEFF2, the reference antibody comprising a heavy chain variable region (VH) of SEQ ID NO:25 and a light chain variable region (VL) of SEQ ID NO:28, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:29, a VH of SEQ ID NO:27 and a VL of SEQ ID NO:30, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:31, a VH of SEQ ID NO:87 and a VL of SEQ ID NO:88, or a VH of SEQ ID NO:89 and a VL of SEQ ID NO:90.

[0009] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane proximal region of TMEFF2, wherein the antibody or the antigen-binding fragment thereof binds to the membrane proximal region of TMEFF2 within residues HGKCEHSINMQEPSC (SEQ ID NO: 57) or DAGYTGQHCEKKDYSVL (SEQ ID NO: 58).

[0010] The present invention also provides an isolated anti-TMEFF2 antibody having certain heavy chain and light chain complementary determining region sequences as described herein.

[0011] The present invention also provides an isolated anti-TMEFF2 antibody having certain heavy chain variable region and light chain variable region sequences as described herein.

[0012] The present invention also provides an isolated anti-TMEFF2 antibody having certain heavy chain and light chain sequences as described herein.

[0013] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0014] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively;

[0015] VH of SEQ ID NO:25 and VL of SEQ ID NO:28; and / or

[0016] HC of SEQ ID NO:32 and LC of SEQ ID NO:35.

[0017] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0018] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively;

[0019] VH of SEQ ID NO:26 and VL of SEQ ID NO:29; and / or

[0020] HC of SEQ ID NO:33 and LC of SEQ ID NO:36.

[0021] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0022] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively;

[0023] VH of SEQ ID NO:27 and VL of SEQ ID NO:30; and / or

[0024] HC of SEQ ID NO:34 and LC of SEQ ID NO:37.

[0025] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0026] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively;

[0027] VH of SEQ ID NO:26 and VL of SEQ ID NO:31; and / or

[0028] HC of SEQ ID NO:33 and LC of SEQ ID NO:38.

[0029] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0030] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively;

[0031] VH of SEQ ID NO:87 and VL of SEQ ID NO:88; and / or

[0032] HC of SEQ ID NO:91 and LC of SEQ ID NO:92.

[0033] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises:

[0034] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively;

[0035] The VH of SEQ ID NO:89 and the VL of SEQ ID NO:90; and / or

[0036] HC of SEQ ID NO:93 and LC of SEQ ID NO:94.

[0037] The present invention also provides a pharmaceutical composition comprising the anti-TMEFF2 antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0038] The present invention also provides an isolated polynucleotide, which encodes the anti-TMEFF2 antibody or its antigen-binding fragment of the present invention, encodes the anti-TMEFF2 antibody VH of SEQ ID NO:25, 26, 27, 87 or 89 and / or the VL of SEQ ID NO:28, 29, 30, 31, 88 or 90, or comprises a polynucleotide sequence of SEQ ID NO:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 95, 96, 97, 98, 99, 100, 101 or 102.

[0039] The present invention also provides a vector comprising the polynucleotide of the present invention.

[0040] The present invention also provides a host cell comprising the vector of the present invention.

[0041] The present invention also provides a method for preparing the anti-TMEFF2 antibody or its antigen-binding fragment of the present invention, which comprises culturing the host cell of the present invention under conditions where the antibody or antigen-binding fragment is expressed, and recovering the antibody or antigen-binding fragment produced by the host cell.

[0042] The present invention also provides a method for treating TMEFF2-positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention to treat TMEFF2-positive cancer.

[0043] The present invention also provides an anti-idiotypic antibody that binds to the anti-TMEFF2 antibody of the present invention.

[0044] The present invention also provides a kit comprising the anti-TMEFF2 antibody or antigen-binding fragment thereof of the present invention.

[0045] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3.

[0046] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the antibody binds to the membrane proximal region of SEQ ID NO: 110 of TMEFF2.

[0047] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody competes with a reference antibody for binding to the membrane proximal region of TMEFF2, the reference antibody comprising a heavy chain variable region (VH) of SEQ ID NO:25 and a light chain variable region (VL) of SEQ ID NO:28, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:29, a VH of SEQ ID NO:27 and a VL of SEQ ID NO:30, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:31, a VH of SEQ ID NO:87 and a VL of SEQ ID NO:88, or a VH of SEQ ID NO:89 and a VL of SEQ ID NO:90.

[0048] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody binds to the epitope of SEQ ID NO: 57 or SEQ ID NO: 58 on TMEFF2.

[0049] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0050] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0051] The first domain comprises the VH of SEQ ID NO:25 and the VL of SEQ ID NO:28, and the second domain comprises the VH of SEQ ID NO:66 and the VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises a first heavy chain (HC1) of SEQ ID NO:32, a first light chain (LC1) of SEQ ID NO:35, a second heavy chain (HC2) of SEQ ID NO:76, and a second light chain (LC2) of SEQ ID NO:77.

[0052] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0053] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0054] The first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:32, LC1 of SEQ ID NO:35, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0055] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0056] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0057] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0058] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0059] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0060] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0061] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0062] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0063] The first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0064] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0065] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0066] The first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0067] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0068] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0069] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0070] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0071] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0072] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0073] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0074] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0075] The first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:91, LC1 of SEQ ID NO:92, HC2 of SEQ ID NO:76 and LC2 of SEQ ID NO:77.

[0076] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0077] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0078] The first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:91, LC1 of SEQ ID NO:92, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0079] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0080] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0081] The first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:93, LC1 of SEQ ID NO:94, HC2 of SEQ ID NO:76 and LC2 of SEQ ID NO:77.

[0082] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0083] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0084] The first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:93, LC1 of SEQ ID NO:94, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0085] The present invention also provides a pharmaceutical composition comprising the bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0086] The present invention also provides a polynucleotide encoding the bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof of the present invention.

[0087] The present invention also provides a method for preparing a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises culturing the host cell of the present invention under conditions that express the antibody, and recovering and purifying the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment produced by the host cell.

[0088] The present invention also provides a method for preparing a bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, the method comprising:

[0089] Mixing a monospecific bivalent TMEFF2 antibody having two identical HC1s and two identical LC1s with a monospecific bivalent CD3 antibody having two identical HC2s and two identical LC2s in a mixture of about a 1:1 molar ratio;

[0090] introducing a reducing agent into the mixture;

[0091] Incubating the mixture for about ninety minutes to about six hours;

[0092] removing the reducing agent; and

[0093] Purify a bispecific anti-TMEFF2 / anti-CD3-antibody or antigen-binding fragment thereof comprising HC1, LC1, HC2 and LC2.

[0094] The present invention also provides a method for treating TMEFF2-positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific anti-TMEFF2 / anti-CD3-antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention to treat TMEFF2-positive cancer.

[0095] The present invention also provides an anti-idiotypic antibody that binds to the bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof of the present invention.

[0096] The present invention also provides a kit comprising the bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 An alignment of selected anti-TMEFF2 antibody heavy chain variable regions (VH) is shown. The VH regions are identified at the beginning of each row by their SEQ ID NO:

[0098] Figure 2 An alignment of selected anti-TMEFF2 antibody light chain variable regions (VL) is shown. The VH regions are identified at the beginning of each row by their SEQ ID NO:

[0099] Figure 3 Shown is the killing of LnCaP cells by selected bispecific anti-TMEFF2 / anti-CD3 antibodies over time as measured by increase in caspase 3 / 7 activity.

[0100] Figure 4 Shown is the reduction in mean tumor volume over time following tumor implantation by selected bispecific anti-TMEFF2 / anti-CD3 antibodies in an ex vivo LnCaP prostate cancer model in male NGS mice.

[0101] Figure 5A Shown is the reduction in mean tumor volume per mouse treated with 0.5 mg / kg TMCB93 in the ex vivo LnCaP prostate cancer model in male NGS mice.

[0102] Figure 5B Shown is the reduction in mean tumor volume per mouse treated with 0.5 mg / kg TMCB132 in an ex vivo LnCaP prostate cancer model in male NGS mice.

[0103] Figure 6 The efficacy of TMEB762xCD3B376 in established LNCaP xenografts in T cell humanized NSG mice is shown.

[0104] Figure 7 Shown is T cell activation in LnCaP prostate cancer cells in response to administration of TMCB132.

[0105] Figure 8 T cell-mediated cytotoxicity of TMCB132 is shown.

[0106] Fig. 9 The anti-tumor efficacy of TMCB132 in T cell humanized mice is shown. DETAILED DESCRIPTION

[0107] All publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if fully set forth herein.

[0108] It should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0109] Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, exemplary materials and methods are described herein.In describing and claiming the present invention, the following terminology will be used.

[0110] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and so forth.

[0111] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprises," "comprising," and the like should be construed in an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0112] "Specific binding", "specifically binds" or "binding" means that the antibody binds to an antigen or an epitope within an antigen with a higher affinity than for other antigens. -8 M or less (e.g., about 1×10 -9 M or smaller, about 1×10 -10 M or smaller, about 1×10 -11 M or smaller, or about 1×10 -12 M or less) D ) binds to an antigen or an epitope within an antigen, typically the K D is the K of the antibody binding to non-specific antigens (such as BSA, casein) DAt least one percent of the dissociation constant. The dissociation constant can be measured using the protocols described herein. However, an antibody that binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous) (such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp). A monospecific antibody binds to one antigen or one epitope, while a bispecific antibody binds to two different antigens or two different epitopes.

[0113] "Antibody" refers to and includes immunoglobulin molecules in a broad sense, including monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetraspecific antibodies, etc.), dimers, tetramers or multimers, single-chain antibodies, domain antibodies, and any other modified configurations of immunoglobulin molecules containing antigen-binding sites with desired specificity. "Full-length antibodies" contain two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds and their multimers (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH region and the VL region can be further subdivided into hypervariable regions, which are called complementarity determining regions (CDRs) and are interspersed with framework regions (FRs). Each VH and VL is composed of three CDR and four FR segments, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0114] "Complementarity determining region (CDR)" is the region of an antibody that binds to an antigen. CDRs can be defined using various delineations, such as Kabat (Wu et al., (1970) J Exp Med 132:211-50) (Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton, (1996) J Bmol Biol 263:800-15). The correspondence between various delineations and variable region numbers is described (see, e.g., Lefranc et al., (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, (2001) J Mol Biol 309:657-70; International Immunogenetics (IMGT) database; Web resource, www.imgt.org). Available programs (such as abYsis of UCL Business PLC) can be used to delineate CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods (Kabat, Chothia, IMGT or AbM).

[0115] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, kappa and lambda, based on the amino acid sequence of their constant domains.

[0116] "Antigen binding fragment" refers to the part of an immunoglobulin molecule that binds to an antigen. Antigen binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides, and include VH, VL, VH and VL, Fab, F(ab')2, Fd and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of antibodies (such as FR3-CDR3-FR4 portions, HCDR1, HCDR2 and / or HCDR3, and LCDR1, LCDR2 and / or LCDR3). The VH and VL domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, in which the VH and VL domains are expressed by separate single-chain antibody constructs, and the VH / VL domains can pair intramolecularly or intermolecularly to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or a diabody; for example, as described in International Patent Publications WO1998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO1992 / 01047.

[0117] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known changes (such as removal of the C-terminal lysine from the antibody heavy chain) or post-translational modifications (such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation). Monoclonal antibodies generally bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific monovalent, bivalent or multivalent.

[0118] "Isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or proteins, such as antibodies) that have been substantially separated and / or purified from other components of a system (such as a recombinant cell) in which the molecules were produced, as well as proteins that have been subjected to at least one purification or separation step. "Isolated antibody" refers to an antibody that is substantially free of other cellular material and / or chemicals, and encompasses antibodies isolated to a higher degree of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0119] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. A humanized antibody may comprise substitutions in the framework so that the framework may not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0120] "Human antibody" refers to an antibody optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is derived from a system using human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region and a light chain variable region "derived from" a sequence of human origin. Such exemplary systems are human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Due to the differences between the systems for obtaining human antibodies and human immunoglobulin loci, the introduction of somatic mutations, or the intentional introduction of substitutions into a framework or CDR, or both, "human antibodies" generally comprise amino acid differences compared to immunoglobulins expressed in humans. Typically, the amino acid sequence of a "human antibody" has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may include a consensus framework sequence obtained by human framework sequence analysis (e.g., Knappik et al., (2000) J Mol Biol 296: described in 57-86); or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on a phage (e.g., Shi et al., (2010) J Mol Biol 397: described in 385-96 and International Patent Publication Wo 2009 / 085462).

[0121] The definition of "human antibody" does not include antibodies in which at least one CDR is derived from a non-human species.

[0122] "Recombinant" refers to DNA, antibodies and other proteins that are prepared, expressed, formed or isolated by recombinant means when fragments from different sources are joined to produce a recombinant DNA, antibody or protein.

[0123] "Epitope" refers to the part of an antigen that specifically binds to an antibody. An epitope is usually composed of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of a portion such as an amino acid or a polysaccharide side chain, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be composed of continuous and / or discontinuous amino acids that form a conformational space unit. For a discontinuous epitope, amino acids from different parts of the linear sequence of an antigen are close in three-dimensional space due to the folding of the protein molecule.

[0124] "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific antibodies may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous) (such as humans or monkeys, e.g., macaques (Macaca cynomolgus) or chimpanzees), or may bind to epitopes shared between two or more different antigens.

[0125] "Multispecific" refers to antibodies that specifically bind to two or more different antigens or two or more different epitopes within the same antigen. Multispecific antibodies may have cross-reactivity to other related antigens, for example, to the same antigen from other species (homologous) (such as humans or monkeys, e.g., macaques or chimpanzees), or may bind to epitopes shared between two or more different antigens.

[0126] A "variant" is a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications (eg, one or more substitutions, insertions, or deletions).

[0127] "Vector" refers to a polynucleotide that can replicate within a biological system or can be moved between such systems. Vector polynucleotides usually contain elements such as replication origins, polyadenylation signals or selection markers, whose function is to promote the replication or maintenance of these polynucleotides in biological systems, such as cells, viruses, animals, plants, and recombinant biological systems that utilize biological components capable of replicating vectors. Vector polynucleotides can be single-stranded or double-stranded DNA or RNA molecules or hybrid molecules of these molecules.

[0128] "Expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0129] "Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked to a phosphate sugar backbone or other equivalent covalent chemicals. cDNA is an exemplary synthetic polynucleotide.

[0130] "Polypeptide" or "protein" refers to a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides."

[0131] "TMEFF2" refers to a human transmembrane protein with an EGF-like and two follistatin-like domains 2, also known as tomoregulin 2. The amino acid sequence of full-length human TMEFF2 is shown in SEQ ID NO: 1. The extracellular domain of TMEFF2 is shown in SEQ ID NO: 2 and spans residues 40-374 of full-length TMEFF2. The TMEFF2 extracellular domain carries three different subdomains: Kazal-like 1 (residues 85-137), Kazal-like 2 (residues 176-229) and EGF domain (residues 261-301). The TMEFF2EGF domain is shown in SEQ ID NO: 3. The "membrane proximal region" of TMEFF2 refers to the TMEFF2 region of SEQ ID NO: 110, which covers the EGF domain and the NC-terminal joint region (e.g., residues 230-320 of the full-length human TMEFF2 of SEQ ID NO: 1). Unless explicitly indicated as being from a non-human species, all references herein to proteins, polypeptides and protein fragments are intended to refer to the human form of the corresponding protein, polypeptide or protein fragment. Therefore, unless indicated as being from a non-human species, such as "mouse TMEFF2" or "monkey TMEFF2", "TMEFF2" means human TMEFF2.

[0132] SEQ ID NO: 1 (full length human TMEFF2)

[0133] MVLWESPRQCSSWTLCEGFCWLLLLLPVMLLIVARPVKLAAFPTSLSDCQTPTGWNCSGYD

[0134] DRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYVPVCGSNGESYQNECYLRQAA

[0135] CKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETSTCDICQFGAECDEDAEDVWC

[0136] VCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIEVMSLGRCQDNTTTTTKSEDG

[0137] HYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHC

[0138] EKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGH

[0139] YSSDNTTRASTRLI

[0140] SEQ ID NO: 2 (extracellular domain of human TMEFF2)

[0141] FPTSLSDCQTPTGWNCSGYDDRENDLFLCDTNTCKFDGECLRIGDTVTCVCQFKCNNDYV

[0142] PVCGSNGESYQNECYLRQAACKQQSEILVVSEGSCATDAGSGSGDGVHEGSGETSQKETS

[0143] TCDICQFGAECDEDAEDVWCVCNIDCSQTNFNPLCASDGKSYDNACQIKEASCQKQEKIE

[0144] VMSLGRCQDNTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCM

[0145] HGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYVLIAAVIGTIQIAVICVVVLCITRKCPRSNRIHRQKQNTGHYSSDNTTRASTRLI

[0146] TMEFF2 EGF domain SEQ ID NO: 3

[0147] HHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCE

[0148] TMEFF2 membrane proximal region SEQ ID NO: 110

[0149] NTTTTTKSEDGHYARTDYAENANKLEESAREHHIPCPEHYNGFCMHGKCEHSINMQEPSCRCDAGYTGQHCEKKDYSVLYVVPGPVRFQYV

[0150] "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) complex and composed of homodimers or heterodimers, which are formed by the association of two or four receptor chains: CD3ε, CD3δ, CD3ζ and CD3γ. Human CD3ε comprises the amino acid sequence of SEQ ID NO:4. Unless explicitly indicated as being from a non-human species, all references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide or protein fragment. Therefore, unless indicated as being from a non-human species, such as "mouse CD3" or "monkey CD3", etc., "CD3" means human CD3.

[0151] SEQ ID NO:4 (human CD3ε)

[0152] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQ

[0153] HNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCE

[0154] NCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERP

[0155] PPVPNPDYEPIRKGQRDLYSGLNQRRI

[0156] SEQ ID NO: 5 (human CD3ε extracellular domain)

[0157] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL

[0158] SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0159] “Bispecific anti-TMEFF2 / anti-CD3 antibody,” TMEFF2 / CD3 antibody, TMEFF2xCD3 antibody, and the like refer to antibodies that bind to TMEFF2 and CD3.

[0160] "In combination with" means that two or more therapeutic agents are administered to the subject together as a mixture, simultaneously as single agents, or sequentially in any order as single agents.

[0161] "TMEFF2-positive cancer" refers to cancer tissue or cancer cells that exhibit measurable levels of TMEFF2 protein. The level of TMEFF2 protein can be measured using well-known assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay, on live or lysed cells.

[0162] "Sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluids, tears, excretions, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids of the pleura, pericardium, peritoneum, abdominal cavity, and other body cavities, fluids collected by bronchial lavage, liquid solutions in contact with a subject or biological source such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy specimens, fine needle aspiration or surgically removed tumor tissue.

[0163] "Cancer cell" or "tumor cell" refers to a cancerous, precancerous or transformed cell in vivo, in vitro or in tissue culture, which has spontaneous or induced phenotypic changes. These changes may not necessarily involve the uptake of new genetic material. Although conversion can occur by infection with transforming viruses and in combination with new genomic nucleic acids or ingestion of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Conversion / cancer examples are morphological changes, cell immortality, abnormal growth control, lesion formation, proliferation, malignant tumors, tumor-specific marker level regulation, invasion, tumor growth (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd edition, 1994) in vitro, in vivo and in vitro in suitable animal hosts (such as nude mice, etc.).

[0164] "About" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the examples or elsewhere in the specification, "about" means within one standard deviation, or up to a range of 5%, whichever is greater, according to customary practice in the art.

[0165] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably.

[0166] "Treatment" refers to both therapeutic treatment and preventive or defensive measures, wherein the goal is to prevent or slow down (mitigate) undesirable physiological changes or disorders. Beneficial or desired clinical outcomes include alleviation of symptoms, attenuation of disease extent, stable (i.e., non-exacerbated) state of disease, delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival compared to the expected survival of the subject if he or she is not receiving treatment. Individuals in need of treatment include individuals who already have a disease or disorder and individuals who are susceptible to a disease or disorder or individuals in whom a disease or disorder is to be prevented.

[0167] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and time period. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improving the health of the patient.

[0168] Unless specifically stated otherwise, throughout the specification, amino acid residues in the antibody constant region are numbered according to the EU index as described in Kabat et al., "Sequences of Proteins of Immunological Interest", 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0169] The conventional one-letter and three-letter amino acid codes as shown in Table 1 are used herein.

[0170] Table 1.

[0171] Amino Acids Three letter code Single letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Gln E Glutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0172] Composition of matter

[0173] The present invention provides an anti-TMEFF2 antibody or an antigen-binding fragment thereof, a multispecific antibody comprising an antigen-binding fragment of the anti-TMEFF2 antibody of the present invention, and a bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof. The present invention provides polypeptides and polynucleotides encoding the antibodies of the present invention or their complementary nucleic acids, vectors, host cells, and methods for preparing and using the same.

[0174] Anti-TMEFF2 Antibody

[0175] Relative to other normal tissues, prostate tissue and prostate adenocarcinoma are significantly enriched in TMEFF2 expression. Membrane TMEFF2 is retained throughout the disease progression and is used as a possible target for anti-tumor therapeutic agents. It is known that TMEFF2 is cleaved by proteases to produce a soluble form of the antigen. In the present invention, antibodies against the membrane proximal region of TMEFF2 are produced to maximize the binding of antibodies to membrane TMEFF2 and minimize the possibility of the resulting antibodies binding to soluble TMEFF2 forms.

[0176] The present invention provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, which binds to the membrane proximal region of SEQ ID NO: 110 of TMEFF2. The anti-TMEFF2 antibody of the present invention that binds to the membrane proximal region of TMEFF2 is not internalized by cells. Although not wishing to be bound by any particular theory, it can be expected that, compared to internalizing anti-TMEFF2 antibodies, non-internalizing anti-TMEFF2 antibodies have an improved oncogenic effect mediated by antibody effector function due to lack of internalization and degradation of TMEFF2.

[0177] "Bound to the membrane proximal region" means that 90% of the antibody epitope residues identified using hydrogen / deuterium exchange (H / D exchange) are located within the membrane proximal region of TMEFF2. The epitope residues are those protected by the test antibody with at least 5% difference in deuteration levels by H / D exchange. Exemplary such antibodies are TMEB675, TMEB570, TMEB674, TMEB565, TMEB762, and TMEB757 as described herein.

[0178] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof binds to the membrane proximal region of TMEFF2 within residues HGKCEHSINMQEPSC (SEQ ID NO: 57) or DAGYTGQHCEKKDYSVL (SEQ ID NO: 58). An exemplary anti-TMEFF2 antibody that binds within residues HGKCEHSINMQEPSC (SEQ ID NO: 57) is TMEB570. An exemplary anti-TMEFF2 antibody that binds within residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 58) is TMEB675. TMEB675 variants TMEB762 and TMEB757 are also expected to bind to the membrane proximal region of TMEFF2 within residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 58).

[0179] In the H / D exchange assay, the recombinantly expressed TMEFF2 ECD is incubated in deuterated water for a predetermined time in the presence or absence of an antibody, resulting in the introduction of deuterium at exchangeable hydrogen atoms not protected by the antibody, followed by protease digestion of the protein and analysis of the peptide fragments using LC-MS. The H / D exchange assay can be performed using known protocols. An exemplary protocol is described in Example 5.

[0180] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to the membrane-proximal region of TMEFF2, the reference antibody comprising a heavy chain variable region (VH) of SEQ ID NO:25 and a light chain variable region (VL) of SEQ ID NO:28, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:29, a VH of SEQ ID NO:27 and a VL of SEQ ID NO:30, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:31, a VH of SEQ ID NO:87 and a VL of SEQ ID NO:88, or a VH of SEQ ID NO:89 and a VL of SEQ ID NO:90.

[0181] Competition between a test antibody and a reference antibody for binding to the membrane proximal region of TMEFF2 can be determined in vitro using well-known methods. For example, the binding of an MSD Sulfo-Tag in the presence of an unlabeled reference antibody can be assessed by ELISA. TM The binding of the test antibody labeled with NHS-ester to the proximal region of the TMEFF2 membrane, or competition can be demonstrated using Bioacore analysis or flow cytometry. When the test antibody inhibits the binding of the reference antibody to the proximal region of the membrane of TMEFF2 by 85% or more, such as 90% or more, or 95% or more, the test antibody competes with the reference antibody for binding to TMEFF2.

[0182] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the following heavy chain complementary determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementary determining region 1 (LCDR1), LCDR2 and LCDR3:

[0183] SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively;

[0184] SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively;

[0185] SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively;

[0186] SEQ ID NO: 11, 13, 16, 18, 20 and 22, respectively; or

[0187] They are SEQ ID NO: 10, 12, 15, 18, 20 and 86 respectively.

[0188] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof of the invention is present at about 0.4×10 -9 M or less equilibrium dissociation constant (K D ) binds to the membrane proximal region of TMEFF2, where K D Measurements were performed using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

[0189] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is present in an amount between about 0.1×10 - 10 M and about 0.4×10 -9 K between M D Binds to the membrane-proximal region of TMEFF2.

[0190] The affinity of the antibody to the membrane proximal region of TMEFF2 can be determined experimentally using any suitable method. An exemplary method uses ProteOn XPR36, Biacore 3000 or KinExA instruments, ELISA or competitive binding assays known to those skilled in the art. If measured under different conditions (e.g., osmotic pressure, pH), the affinity of the measured antibody to TMEFF2 can vary. Therefore, affinity and other binding parameters (e.g., K D , K on and K off ) are typically performed using standardized conditions and standardized buffers (such as those described herein). One skilled in the art will appreciate that the internal error (measured as standard deviation, SD) of affinity measurements using, for example, Biacore 3000 or ProteOn can typically be within 5%-33% of the measured value within typical detection limits. Thus, when referring to K D When using values, the term "about" reflects the typical standard deviation in the measurement. For example, 1×10 -9 M of K D The typical SD is at most +0.33×10 -9 M.

[0191] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane proximal region of TMEFF2 and comprises a heavy chain variable region (VH) framework derived from VH3_3-23 (SEQ ID NO: 53) or VH1_1-69 (SEQ ID NO: 54).

[0192] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane proximal region of TMEFF2 and comprises a light chain variable region (VL) framework derived from VKI_L11 (SEQ ID NO: 55) or VKIIII_A27 (SEQ ID NO: 56).

[0193] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane-proximal region of TMEFF2 and comprises a VH framework and a VL framework derived from VH3_3-23 of SEQ ID NO:53 and VKI_L11 of SEQ ID NO:55, respectively.

[0194] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane-proximal region of TMEFF2 and comprises a VH framework and a VL framework derived from VH1_1-69 of SEQ ID NO:54 and VKIII_A27 of SEQ ID NO:56, respectively.

[0195] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which binds to the membrane-proximal region of TMEFF2 and comprises a VH framework and a VL framework derived from VH1_1-69 of SEQ ID NO:54 and VKI_L11 of SEQ ID NO:55, respectively.

[0196] An antibody comprising a heavy or light chain variable region "derived from" a particular framework or germline sequence refers to an antibody obtained from a system that uses human germline immunoglobulin genes, such as from a transgenic mouse, rat, or chicken, or a phage display library as discussed herein. An antibody containing a particular framework derived from a germline sequence may contain amino acid differences compared to the sequence from which it was derived, due to, for example, naturally occurring somatic mutations or deliberate substitutions.

[0197] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively.

[0198] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:25 and the VL of SEQ ID NO:28.

[0199] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:39, and VL is encoded by the polynucleotide of SEQ ID NO:42.

[0200] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:32 and the LC of SEQ ID NO:35.

[0201] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:46, and the VL is encoded by the polynucleotide of SEQ ID NO:49.

[0202] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively.

[0203] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:26 and the VL of SEQ ID NO:29.

[0204] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO:40, and the VL is encoded by the polynucleotide of SEQ ID NO:43.

[0205] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:33 and the LC of SEQ ID NO:36.

[0206] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:47, and the LC is encoded by the polynucleotide of SEQ ID NO:50.

[0207] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10, 14, 17, 18, 20 and 24, respectively.

[0208] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:27 and the VL of SEQ ID NO:30.

[0209] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO:41 and the VL is encoded by the polynucleotide of SEQ ID NO:44.

[0210] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:34 and the LC of SEQ ID NO:37.

[0211] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:48, and the LC is encoded by the polynucleotide of SEQ ID NO:51.

[0212] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively.

[0213] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:26 and the VL of SEQ ID NO:31.

[0214] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO:40, and the VL is encoded by the polynucleotide of SEQ ID NO:45.

[0215] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:33 and the LC of SEQ ID NO:38.

[0216] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:47, and the LC is encoded by the polynucleotide of SEQ ID NO:52.

[0217] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10, 12, 15, 18, 20 and 86, respectively.

[0218] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:87 and the VL of SEQ ID NO:88.

[0219] In some embodiments, the VH is encoded by the polynucleotide of SEQ ID NO:95 and the VL is encoded by the polynucleotide of SEQ ID NO:96.

[0220] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:91 and the LC of SEQ ID NO:92.

[0221] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:97, and the LC is encoded by the polynucleotide of SEQ ID NO:98.

[0222] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10, 12, 15, 18, 20 and 86, respectively.

[0223] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the VH of SEQ ID NO:89 and the VL of SEQ ID NO:90.

[0224] In some embodiments, VH is encoded by the polynucleotide of SEQ ID NO:99 and VL is encoded by the polynucleotide of SEQ ID NO:100.

[0225] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:93 and the LC of SEQ ID NO:94.

[0226] In some embodiments, the HC is encoded by the polynucleotide of SEQ ID NO:101 and the LC is encoded by the polynucleotide of SEQ ID NO:102.

[0227] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is a multispecific antibody.

[0228] In some embodiments, the isolated anti-TMEFF2 antibody or antigen-binding fragment thereof is a bispecific antibody.

[0229] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds a T cell antigen.

[0230] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds CD3.

[0231] In some embodiments, the isolated anti-TMEFF2 bispecific antibody or antigen-binding fragment thereof binds CD3ε.

[0232] The VH, VL, HCDR, LCDR, HC and LC sequences of exemplary anti-TMEFF2 antibodies of the invention are shown in Tables 5-12.

[0233] Although the embodiments shown in the examples include paired variable domains, one from the heavy chain and one from the light chain, it will be appreciated by those skilled in the art that alternative embodiments may include a single heavy chain variable domain or a single light chain variable domain. A single variable domain can be used to screen for variable domains that can form a two-domain specific antigen-binding fragment that can bind to TMEFF2. Screening can be accomplished by phage display screening methods, using, for example, the hierarchical dual combination method disclosed in International Patent Publication WO1992 / 01047. In this method, a single bacterium colony containing a VH or VL chain clone is used to infect a complete library of clones encoding another chain (VL or VH), and the resulting double-chain specific antigen-binding domains are selected according to phage display technology using known methods and those described herein. Therefore, using the method disclosed in International Patent Publication WO1992 / 01047, separate VH and VL polypeptide chains can be used to identify additional anti-TMEFF2 antibodies.

[0234] Homologous antibody

[0235] Variants of the anti-TMEFF2 antibodies of the present invention or their antigen-binding fragments comprising the VH or VL amino acid sequences shown in Table 9 are within the scope of the present invention. For example, the variant may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions in VH and / or VL, as long as the homologous antibody maintains or has improved functional properties (such as comparable or improved stability in binding to TMEFF2) when compared to the parent antibody. In some embodiments, the sequence identity to the VH or VL amino acid sequences of the present invention may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0236] In some embodiments, the homologous anti-TMEFF2 antibody or antigen-binding fragment thereof of the present invention is present at about 0.4×10 -9 M or less equilibrium dissociation constant (K D ) binds to the membrane proximal region of TMEFF2, where K D Measurements were performed using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

[0237] In some embodiments, the homologous anti-TMEFF2 antibody or antigen-binding fragment thereof of the present invention is present in an amount between about 0.1×10 -10 M and about 0.4×10 -9 K between M D Binds to the membrane-proximal region of TMEFF2.

[0238] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising VH of SEQ ID NO: 25 and VL of SEQ ID NO: 28, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitution is not within a CDR.

[0239] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 29, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitution is not within a CDR.

[0240] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising VH of SEQ ID NO: 27 and VL of SEQ ID NO: 30, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitution is not within a CDR.

[0241] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 31, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitution is not within a CDR.

[0242] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising VH of SEQ ID NO: 87 and VL of SEQ ID NO: 88, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitution is not within a CDR.

[0243] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising VH of SEQ ID NO: 89 and VL of SEQ ID NO: 90, wherein VH, VL, or both VH and VL optionally comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. Optionally, any substitution is not within a CDR.

[0244] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the VH of SEQ ID NO: 25, 26, 27, 87 or 89. Optionally, any variation in the sequence of SEQ ID NO is not within the CDR.

[0245] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the VL of SEQ ID NO: 28, 29, 30, 31, 88 or 90. Optionally, any variation in the sequence of SEQ ID NO is not within the CDR.

[0246] The amino acid sequence alignment of the VH domain of the anti-TMEFF2 antibody is shown in Figure 1 , and the alignment of the amino acid sequences of the VL domains of selected anti-TMEFF2 antibodies is shown in Figure 2 In. VH chain and VL chain are marked by their SEQ ID NO: at the beginning of each row. Possible substitution sites in VH and / or VL are different residue positions between each antibody. For example, in the VH of SEQ ID NO:25,27,87 and 89 (numbered based on SEQ ID NO 25), substitutions can be made at residue positions 14,20,54,56,59,76,82,84,93,107. Similarly, in the VL of SEQ ID NO:28,30,88 and 90, substitutions can be made at residue positions 1,95 and 107. Exemplary substitutions that can be made are conservative amino acid substitutions, or substitutions made with the amino acid residues present in the corresponding residue positions in each anti-TMEFF2 antibody.

[0247] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which parameters need to be introduced for optimal alignment of the two sequences.

[0248] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J Mol Biol 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0249] Antibodies with conservative modifications

[0250] The present invention also provides an isolated anti-TMEFF2 antibody or antigen-binding fragment thereof, comprising a VH comprising HCDR1, HCDR2 and HCDR3 sequences and a VL comprising LCDR1, LCDR2 and LCDR3 sequences, wherein one or more of the CDR sequences comprises a specific amino acid sequence based on an antibody described herein (e.g., an antibody shown in Tables 5-12) or a conservative modification thereof, and wherein the antibody retains the desired functional properties of the parent antibody.

[0251] In some embodiments, the conservatively modified anti-TMEFF2 antibody or antigen-binding fragment thereof is present at about 0.4×10 -9 M or less equilibrium dissociation constant (K D ) binds to the membrane proximal region of TMEFF2, where K D Measurements were performed using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

[0252] In some embodiments, the conservatively modified anti-TMEFF2 antibody or antigen-binding fragment thereof is present in an amount between about 0.1×10 -10 M and about 0.4×10 -9 K between M D Binds to the membrane-proximal region of TMEFF2.

[0253] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10, 12, 15, 18, 20 and 22, respectively, and conservative modifications thereof.

[0254] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 11, 13, 16, 19, 21 and 23, respectively, and conservative modifications thereof.

[0255] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10, 14, 17, 18, 20 and 24, respectively, and conservative modifications thereof.

[0256] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 11, 13, 16, 18, 20 and 22, respectively, and conservative modifications thereof.

[0257] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 10, 12, 15, 18, 20 and 86, respectively, and conservative modifications thereof.

[0258] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 28 and conservative modifications thereof.

[0259] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 29 and conservative modifications thereof.

[0260] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the VH of SEQ ID NO: 27 and the VL of SEQ ID NO: 30 and conservative modifications thereof.

[0261] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 31 and conservative modifications thereof.

[0262] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the VH of SEQ ID NO: 87 and the VL of SEQ ID NO: 88 and conservative modifications thereof.

[0263] The present invention also provides an isolated anti-TMEFF2 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises the VH of SEQ ID NO: 89 and the VL of SEQ ID NO: 90 and conservative modifications thereof.

[0264] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. "Conservative amino acid substitutions" are substitutions in which an amino acid is replaced by an amino acid residue with a similar side chain. Families of amino acid residues having similar side chains are well defined and include amino acids having the following side chains: acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). In addition, any native residue in the polypeptide can also be replaced with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; ​​Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies of the invention can be performed by known methods, such as by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, a library of variants can be generated, for example, using random (NNK) or non-random codons (e.g., DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The characteristics of the resulting antibody variants can be tested using the assays described herein.

[0265] Immunoconjugates

[0266] An "immunoconjugate" refers to an antibody of the invention conjugated to one or more heterologous molecules.

[0267] The present invention also provides an immunoconjugate comprising the isolated antibody or antigen-binding fragment thereof of the present invention conjugated to a heterologous molecule.

[0268] In some embodiments, the heterologous protein is a detectable tag.

[0269] The isolated antibody or antigen-binding fragment thereof of the present invention conjugated to a detectable label can be used to assess the expression of TMEFF2 on a variety of samples. The detectable label includes a composition that enables the isolated antibody or antigen-binding fragment thereof of the present invention to be detected by spectral, photochemical, biochemical, immunochemical or chemical means when conjugated to the isolated antibody or antigen-binding fragment thereof of the present invention.

[0270] Exemplary detectable labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescence quenchers, colored molecules, radioisotopes, scintillators, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tag, myc tag, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors / acceptors, acridinium esters, and colorimetric substrates.

[0271] The detectable label can emit a signal spontaneously, for example, when the detectable label is a radioisotope. In other cases, the detectable label emits a signal after being stimulated by an external field.

[0272] Exemplary radioisotopes may be gamma-emitting, Auger-emitting, beta-emitting, alpha-emitting, or positron-emitting radioisotopes. Exemplary radioisotopes include 3 H. 11 C. 13 C. 15 N. 18 F. 19 F. 55 Co. 57 Co. 60 Co. 61 Cu, 62 Cu, 64 Cu, 67 Cu, 68 Ga, 72 As, 75 Br, 86 Y. 89 Zr, 90 Sr. 94m Tc, 99m Tc, 115 In, 123 1. 124 1. 125 I. 131 1. 211 At 212 Bi, 213 Bi,223 Ra, 226 Ra, 225 Ac and 227 Ac.

[0273] Exemplary metal atoms are metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms , hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium.

[0274] Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

[0275] Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g. Cy3, Cy5, Cy5.5), Alexa Fluors (e.g. Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

[0276] An isolated antibody or antigen-binding fragment thereof of the invention conjugated to a detectable label can be used as an imaging agent.

[0277] The isolated antibody or antigen-binding fragment thereof of the present invention can be conjugated to a detectable label using known methods.

[0278] In some embodiments, the detectable label is complexed with a chelator.

[0279] In some embodiments, the detectable label is conjugated to the antibody or antigen-binding fragment thereof of the invention via a linker.

[0280] Known methods can be used to directly or indirectly connect the detectable label to the antibody or antigen-binding fragment thereof of the present invention. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succinimidyl benzoate; dodecaborate), chelating moieties of both macrocyclic chelators and acyclic chelators, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid ( The peptide linkers include derivatives of TETA, N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiophene (IT), bifunctional derivatives of imidates (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

[0281] Bispecific anti-TMEFF2 / anti-CD3 antibody

[0282] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3.

[0283] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, the antibody or its antigen-binding fragment comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the antibody binds to the membrane proximal region of TMEFF2. Although not wishing to be bound by any particular theory, bispecific antibodies that bind to the membrane proximal region of TMEFF2 can more effectively mediate T-cell-mediated tumor cell killing.

[0284] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the antibody competes with a reference antibody for binding to the membrane proximal region of TMEFF2, the reference antibody comprising a heavy chain variable region (VH) of SEQ ID NO:25 and a light chain variable region (VL) of SEQ ID NO:28, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:29, a VH of SEQ ID NO:27 and a VL of SEQ ID NO:30, a VH of SEQ ID NO:26 and a VL of SEQ ID NO:31, a VH of SEQ ID NO:87 and a VL of SEQ ID NO:88, or a VH of SEQ ID NO:89 and a VL of SEQ ID NO:90.

[0285] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is present at about 0.4×10 -9 M or less dissociation constant (K D ) binds to the membrane proximal region of TMEFF2, where K D Measurements were performed using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

[0286] In some embodiments, the isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof is present in an amount between about 0.1×10 -10 M and about 0.4×10 -9 K between M D Binds to the membrane proximal region of TMEFF2.

[0287] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the first domain comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively; SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively; SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively;

[0288] SEQ ID NO: 11, 13, 16, 18, 20 and 22, respectively; or

[0289] They are SEQ ID NO: 10, 12, 15, 18, 20 and 86 respectively.

[0290] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the second domain comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3:

[0291] SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively; or

[0292] They are SEQ ID NO: 68, 69, 70, 71, 72 and 73 respectively.

[0293] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the first domain comprises

[0294] VH of SEQ ID NO:25 and VL of SEQ ID NO:28;

[0295] VH of SEQ ID NO:26 and VL of SEQ ID NO:29;

[0296] VH of SEQ ID NO:27 and VL of SEQ ID NO:30;

[0297] VH of SEQ ID NO:26 and VL of SEQ ID NO:31;

[0298] The VH of SEQ ID NO:87 and the VL of SEQ ID NO:88; or

[0299] VH of SEQ ID NO:89 and VL of SEQ ID NO:90.

[0300] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof, wherein the second domain comprises

[0301] The VH of SEQ ID NO:66 and the VL of SEQ ID NO:67; or

[0302] VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0303] In some embodiments, the second domain comprises the VH of SEQ ID NO:59 and the VL of SEQ ID NO:111.

[0304] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0305] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0306] The first domain comprises the VH of SEQ ID NO:25 and the VL of SEQ ID NO:28, and the second domain comprises the VH of SEQ ID NO:66 and the VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises a first heavy chain (HC1) of SEQ ID NO:32, a first light chain (LC1) of SEQ ID NO:35, a second heavy chain (HC2) of SEQ ID NO:76, and a second light chain (LC2) of SEQ ID NO:77.

[0307] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0308] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0309] The first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:32, LC1 of SEQ ID NO:35, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0310] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0311] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0312] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0313] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0314] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0315] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0316] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0317] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0318] The first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0319] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0320] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0321] The first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0322] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0323] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0324] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0325] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0326] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0327] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0328] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0329] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0330] The first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises a first heavy chain (HC1) of SEQ ID NO:91, a first light chain (LC1) of SEQ ID NO:92, a second heavy chain (HC2) of SEQ ID NO:76, and a second light chain (LC2) of SEQ ID NO:77.

[0331] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0332] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0333] The first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:91, LC1 of SEQ ID NO:92, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0334] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0335] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0336] The first domain comprises the VH of SEQ ID NO:89 and the VL of SEQ ID NO:90, and the second domain comprises the VH of SEQ ID NO:66 and the VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises a first heavy chain (HC1) of SEQ ID NO:93, a first light chain (LC1) of SEQ ID NO:94, a second heavy chain (HC2) of SEQ ID NO:76, and a second light chain (LC2) of SEQ ID NO:77.

[0337] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0338] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0339] The first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:93, LC1 of SEQ ID NO:94, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0340] Engineered and modified antibodies

[0341] The antibodies of the present invention or their antigen-binding fragments can be further engineered to produce modified antibodies having similar or altered properties when compared to the parent antibody. In the antibodies of the present invention, any or all of VH, VL, VH and VL, constant regions, heavy chain frameworks, light chain frameworks, or six CDRs can be engineered.

[0342] The antibodies of the present invention can be engineered by CDR grafting. One or more CDR sequences of the antibodies of the present invention can be grafted to different framework sequences. CDR grafting can be performed using known methods and the methods described herein.

[0343] Useful framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA and encoded protein sequences of human heavy and light chain variable domain genes can be found at ,the international ImMunoGeneTics information www.Imgt.org. The framework sequences that can be used to replace the existing framework sequences of the antibodies of the present invention can be those that show the highest percentage (%) identity with the parent variable domains over the entire length of VH or VL or over the length of FR1, FR2, FR3 and FR4. In addition, suitable frameworks can be further selected based on the length of VH and VL CDR1 and CDR2 or the same LCDR1, LCDR2, LCDR3, HCDR1 and HCDR2 canonical structures. Suitable frameworks can be selected using known methods, such as the human framework modification described in U.S. Patent No. 8,748,356 or the superhumanization of U.S. Patent No. 7,709,226.

[0344] The framework sequence of parent and engineered antibody can be further modified, for example, by back mutation, to restore and / or improve the combination of generated antibody and antigen, as described in, for example, U.S. Patent 6,180,370. The framework sequence of parent or engineered antibody can be further modified by mutating one or more residues in the framework region (or alternatively, in one or more CDR regions) to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. The method is also referred to as "de-immunization", and is further described in detail in U.S. Patent Publication US20070014796.

[0345] The CDR residues of the antibodies of the invention may be mutated to adjust the affinity of the antibody for TMEFF2 and / or CD3.

[0346] The CDR residues of the antibodies of the present invention can be mutated to minimize the risk of post-translational modifications. The amino acid residues of the putative motifs for deamination (NS), acid-catalyzed hydrolysis (DP), isomerization (DS) or oxidation (W) can be replaced with any naturally occurring amino acid to mutagenesis these motifs, and the function and stability of the resulting antibodies can be tested using the methods described herein.

[0347] Antibodies of the invention modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desired biological or biophysical properties are within the scope of the invention. The stability of an antibody is affected by many factors, including (1) core stacking of individual domains that affects its intrinsic stability, (2) protein / protein interface interactions that have an impact on HC and LC pairing, (3) embedding of polar and charged residues, (4) H-bond networks of polar and charged residues; and (5) surface charge and polar residue distribution among other intramolecular and intermolecular forces (Worn et al., (2001) J Mol Biol 305:989-1010). Potentially structurally unstable residues can be identified based on the crystal structure of the antibody or in some cases by molecular modeling, and the effects of the residues on antibody stability can be tested by generating and evaluating variants carrying mutations in the identified residues. One method of increasing antibody stability is to increase the thermal transition midpoint (T midpoint) measured by differential scanning calorimetry (DSC). m ). Generally speaking, protein T m It is related to its stability and negatively correlated with its sensitivity to unfolding and denaturation in solution and to degradation processes that depend on the tendency of the protein to unfold (Remmele et al., (2000) Biopharm 13:36-46). A large number of studies have found correlations between the level of physical stability of formulations measured by DSC as thermal stability and physical stability measured by other methods (Gupta et al., (2003) AAPS Pharm Sci 5E8; Zhang et al., (2004) J Pharm Sci 93:3076-89; Maa et al., (1996) Int J Pharm 140:155-68; Bedu-Addo et al., (2004) Pharm Res 21:1353-61; Remmele et al., (1997) Pharm Res 15:200-8). Formulation studies suggest that Fab T m It has an impact on the long-term physical stability of the corresponding mAb.

[0348] The C-terminal lysine (CTL) can be removed from injected antibodies by endogenous circulating carboxypeptidases in the bloodstream (Cai et al., (2011) Biotechnol Bioeng 108:404-412). During production, the C-terminal lysine (CTL) can be removed by controlling the extracellular Zn 2+ , EDTA or EDTA–Fe 3+ The concentration of CTL is controlled to less than the maximum level, as described in US Patent Publication US20140273092. The CTL content in the antibody can be determined using known methods.

[0349] The antibodies of the present invention may be subjected to Fc substitution to modulate antibody effector functions and / or pharmacokinetic properties. In traditional immune functions, the interaction of antibody-antigen complexes with cells of the immune system produces a variety of responses ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation and phagocytosis to immunomodulatory signals such as regulation of lymphocyte proliferation and antibody secretion. All of these interactions are caused by binding of the Fc domain of the antibody or immune complexes to specific cell surface receptors on cells. The diversity of cellular responses triggered by antibodies and immune complexes is caused by the heterogeneity of the following Fc receptors: FcγRI (CD64), FcγRIIa (CD32A) and FcγRIII (CD16) are activating Fcγ receptors (i.e., immune system enhancement), while FcγRIIB (CD32B) is an inhibitory Fcγ receptor (i.e., immune system suppression). Binding to the FcRn receptor modulates the antibody half-life.

[0350] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention comprises at least one substitution in the Fc region.

[0351] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen substitutions in the Fc region.

[0352] Fc position can be replaced to regulate antibody half-life.Exemplary substitutions that can be used to increase antibody half-life are M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A and T307A / E380A / N434A, respectively.Exemplary substitutions that can be used to reduce antibody half-life are H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A and H435R, respectively.

[0353] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention comprises at least one substitution in the Fc region selected from the group consisting of: M428L / N434S, M252Y / S254T / T256E, T250Q / M428L, N434A, T307A / E380A / N434A, H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R.

[0354] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention comprises at least one substitution in the Fc region that reduces binding of the antibody to an activating Fcγ receptor (FcγR) and / or reduces Fc effector function, such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0355] The Fc positions that reduced binding of the antibody to activating FcγRs and subsequently reduced effector function were the following substitutions: L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on all Ig isotypes, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236-deletion / A327G on IgG1 / P331A / D365E / L358M, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236-deletion / G237A / P238S on IgG4.

[0356] It is well known that the S228P substitution can be made in IgG4 antibodies to enhance IgG4 stability.

[0357] In some embodiments, an anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention comprises an S228P substitution, wherein the residues are numbered according to the EU index.

[0358] In some embodiments, an anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention comprises a F234A, L235A, or F234A / L235A substitution, wherein the residues are numbered according to the EU index.

[0359] In some embodiments, an anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention comprises S228P, F234A, and L235A substitutions, wherein the residues are numbered according to the EU index.

[0360] Methods for producing homologous antibodies, antibodies with conservative modifications, and engineering and modifying antibodies

[0361] Antibodies of the present invention having altered amino acid sequences compared to the parent antibody can be produced using standard cloning and expression techniques. For example, site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and the effects on antibody binding or other properties of interest can be assessed using known methods and the methods described in the Examples herein.

[0362] Antibody Isotypes and Allotypes

[0363] The anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention may be of IgG1, IgG2, IgG3 or IgG4 isotype.

[0364] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the IgG1 isotype.

[0365] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the IgG2 isotype.

[0366] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the IgG3 isotype.

[0367] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the IgG4 isotype.

[0368] The immunogenicity of therapeutic antibodies is associated with an increased risk of infusion reactions and a reduced duration of therapeutic response (Baert et al., (2003) NEngl J Med 348:602-08). The extent to which a therapeutic antibody induces an immune response in a host can be determined in part by the allotype of the antibody (Stickler et al., (2011) Genes and Immunity 12:213-21). Antibody allotypes are associated with amino acid sequence variations at specific positions in the constant region sequence of an antibody. Table 2 shows selected IgG1, IgG2, and IgG4 allotypes.

[0369] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the G2m(n) allotype.

[0370] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the G2m(n-) allotype.

[0371] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the G2m(n) / (n-) allotype.

[0372] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the nG4m(a) allotype.

[0373] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the G1m(17) allotype.

[0374] In some embodiments, the anti-TMEFF2 antibody or bispecific anti-TMEFF2 / anti-CD3 antibody of the invention is of the G1m(17,1) allotype.

[0375] Table 2

[0376]

[0377] Anti-idiotypic antibodies

[0378] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody or the bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention.

[0379] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody of the present invention comprising VH of SEQ ID NO: 25 and VL of SEQ ID NO: 28.

[0380] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody of the present invention comprising VH of SEQ ID NO:26 and VL of SEQ ID NO:29.

[0381] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody of the present invention comprising VH of SEQ ID NO: 27 and VL of SEQ ID NO: 30.

[0382] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody of the present invention comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 31.

[0383] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody of the present invention comprising VH of SEQ ID NO: 87 and VL of SEQ ID NO: 88.

[0384] The present invention also provides an anti-idiotypic antibody that specifically binds to the anti-TMEFF2 antibody of the present invention comprising VH of SEQ ID NO: 89 and VL of SEQ ID NO: 90.

[0385] Anti-idiotypic (Id) antibodies are antibodies that recognize the antigenic determinants (e.g., paratopes or CDRs) of an antibody. Id antibodies can be antigen-blocked or non-blocked. Antigen-blocked Ids can be used to detect free antibodies in a sample (e.g., the anti-TMEFF2 antibodies of the present invention described herein). Non-blocked Ids can be used to detect total antibodies (free, partially bound to the antigen, or completely bound to the antigen) in a sample. Id antibodies can be prepared by immunizing an animal for which the anti-Id is being prepared with the antibody.

[0386] Anti-Id antibodies can also be used as immunogens to induce an immune response in another animal, thereby producing so-called anti-anti-Id antibodies. Anti-anti-Id can be identical in epitope to the original mAb that induced anti-Id. Thus, by using antibodies directed against the idiotypic determinants of the monoclonal antibody, other clones expressing the same specific antibody can be identified. Anti-Id antibodies can be altered (thereby producing anti-Id antibody variants) and / or derived by any suitable technique, such as those described elsewhere herein.

[0387] Generation of monospecific antibodies of the invention

[0388] In some embodiments, the anti-TMEFF2 antibody is human.

[0389] In some embodiments, the anti-TMEFF2 antibody is humanized.

[0390] The monospecific antibodies of the present invention can be produced using various techniques. For example, the hybridoma method described in Kohler and Milstein, Nature 256:495, 1975 can be used to produce monoclonal antibodies. In the hybridoma method, a mouse or other host animal (such as a hamster, rat or monkey) is immunized with human, chimpanzee or macaque TMEFF2 or a fragment of TMEFF2 such as the membrane proximal domain of TMEFF2, and then spleen cells from the immunized animal are fused with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Colonies produced by a single immortalized hybridoma cell are screened for the preparation of antibodies having desired properties (such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, and affinity for the antigen).

[0391] Various host animals can be used to produce the anti-TMEFF2 antibodies of the present invention. For example, Balb / c mice can be used to produce mouse anti-human TMEFF2 antibodies. Various techniques can be used to humanize antibodies prepared by Balb / c mice and other non-human animals to produce more human-like sequences.

[0392] Exemplary humanization techniques including selection of human acceptor frameworks are known and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28:489-499), resurfacing of specificity-determining residues (U.S. Pat. Publication No. 2010 / 0261620), human framework reshaping (U.S. Pat. No. 8,748,356), or superhumanization (U.S. Pat. No. 7,709,226). In these methods, the CDRs of a parent antibody are transferred to a human framework, which can be selected based on its overall homology to the parent framework, based on similarity in CDR length, or canonical structural identity, or a combination thereof.

[0393] The humanized antibodies can be further optimized to improve their selectivity or affinity for the desired antigen by introducing modified framework support residues to maintain binding affinity (back mutations) using techniques such as those described in International Patent Publications WO1090 / 007861 and WO1992 / 22653, or by introducing variants at any CDR to improve the affinity of the antibody.

[0394] Transgenic animals (such as mice, rats or chickens) carrying human immunoglobulin (Ig) loci in their genomes can be used to generate human antibodies against target proteins, as described, for example, in U.S. Pat. No. 6,150,584; International Patent Publication No. WO99 / 45962; International Patent Publications No. WO2002 / 066630, WO2002 / 43478, WO2002 / 043478 and WO1990 / 04036; Lonberg et al. (1994) Nature 368:856-9; Green et al. (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int Rev Immunol 13:65-93; Bruggemann et al. (1991) Eur J Immunol 21:1323-1326; Fishwild et al., (1996) Nat Biotechnol 14:845-851; Mendez et al., (1997) Nat Genet 15:146-156; Green (1999) J Immunol Methods 231:11-23; Yang et al., (1999) Cancer Res 59:1236-1243; Brüggemann and Taussig (1997) Curr Opin Biotechnol 8:455-458. The endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the genome of the animal by homologous or nonhomologous recombination using a transchromosome or minigene. Companies such as Regeneron (www.Regeneron.com), Harbour Antibodies (www.Harbourantibodies.com), Open Monoclonal Technology, Inc. (OMT) (www.Omtinc.net), KyMab (www.Kymab.com), Trianni (www.Trianni.com) and Ablexis (www.Ablexis.com) can be involved to provide human antibodies to a selected antigen using the techniques described above.

[0395] Human antibodies can be selected from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., (2000) J Mol Biol 296:57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan et al., (1996) Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). The antibodies of the invention can be isolated, for example, from a phage display library expressing the antibody heavy and light chain variable regions as fusion proteins using phage pIX coat protein, as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication WO09 / 085462. Phages that bind to human and / or cyno TMEFF2 or CD3 can be screened from the library, and the positive clones obtained can be further characterized, Fabs isolated from the clone lysates, and expressed as full-length IgGs. Such phage display methods for isolating human antibodies are described, for example, in U.S. Patents 5,223,409; 5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081.

[0396] Preparation of immunogenic antigens and generation of monoclonal antibodies can be carried out with any suitable technology such as recombinant protein production. Immunogenic antigens can be administered to animals in the form of purified proteins or protein mixtures (including whole cells or cell extracts or tissue extracts), or the antigens can be generated de novo in animals from nucleic acids encoding the antigens or portions thereof.

[0397] Generation of bispecific anti-TMEFF2 / anti-CD3 antibodies of the invention

[0398] The bispecific anti-TMEFF2 / anti-CD3 antibodies of the present invention can be produced by combining the TMEFF2 VH / VL domains separated herein with any CD3 VH / VL domains, including those described herein and those publicly available. Exemplary CD3 VH / VL domains that can be used are those of antibodies CD3B219 and CD3B376 as described herein. Exemplary TMEFF2 VH / VL domains that can be used are those of antibodies TMEB675, TMEB570, TMEB674, TMEB565, TMEB762, and TMEB757. The generated bispecific anti-TMEFF2 / anti-CD3 antibodies can be tested for binding to TMEFF2 and CD3 and their desired functional characteristics, such as T- cell-mediated killing of cells (e.g., LNCaP) expressing TMEFF2.

[0399] The bispecific antibodies of the present invention can be produced, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies in the following manner: a substitution is introduced at the heavy chain CH3 junction in each half molecule to promote the formation of heterodimers of two antibody half molecules with different specificities in an in vitro cell-free environment or using co-expression. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with a cysteine ​​residue of a second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and re-formed by dissociation-association. The CH3 domain of the Fab arm can be modified to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each of which binds to a different epitope, i.e., an epitope on TMEFF2 and an epitope on CD3. For example, the bispecific antibodies of the present invention can be used as described in International Patent Publication WO2011 / 131746. The invention relates to a method for producing bispecific antibodies. In the case of IgG1 antibodies, a mutation F405L in one heavy chain and K409R in another heavy chain can be used. For IgG2 antibodies, wild-type IgG2 and IgG2 antibodies with F405L and R409K substitutions can be used. For IgG4 antibodies, wild-type IgG4 and IgG4 antibodies with F405L and R409K substitutions can be used. In order to produce bispecific antibodies, the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have the aforementioned mutations in the Fc region, and the antibodies are incubated together under reducing conditions sufficient to allow disulfide isomerization of the cysteine ​​in the hinge region; thereby producing bispecific antibodies by Fab arm exchange. The incubation conditions are most ideally restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine ​​and β-mercaptoethanol. For example, the following conditions can be used: incubation for at least 90 minutes at a pH of 5-8, such as pH 7.0 or pH 7.4, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a temperature of at least 20°C.

[0400] In some embodiments, the bispecific anti-TMEFF2 / anti-CD3 antibody is of the IgG1 isotype and comprises a F405L substitution in the first heavy chain (HC1) and a K409R substitution in the second heavy chain (HC2) when compared to the wild-type IgG1 of SEQ ID NO:84.

[0401] In some embodiments, the bispecific anti-TMEFF2 / anti-CD3 antibody is of the IgG4 isotype and comprises F405L / R409K substitutions in HC2 when compared to the wild-type IgG4 of SEQ ID NO:85.

[0402] In some embodiments, the bispecific anti-TMEFF2 / anti-CD3 antibody is of the IgG4 isotype and comprises S228P, F234A, and L235A substitutions in HC1 and S228P, F234A, L235A, F405L, and R409K substitutions in HC2 when compared to the wild-type IgG4 of SEQ ID NO:85.

[0403] SEQ ID NO:84 Wild-type IgG1

[0404] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0405] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0406] PSVFLFPKPKDTLMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0407] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

[0408] LTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

[0409] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0410] SEQ ID NO:85 Wild-type IgG4

[0411] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0412] GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV

[0413] FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

[0414] RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSIEKTISKAKGQPREPQVYTLPPSQEEMTK

[0415] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG

[0416] NVFSCSVMHEALHNHYTQKSLSLSLGK

[0417] SEQ ID NO 103: IgG1 F405L

[0418] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0419] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0420] PSVFLFPKPKDTLMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0421] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

[0422] LTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRW

[0423] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0424] SEQ ID NO:109:IgG1 K409R

[0425] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0426] GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG

[0427] PSVFLFPKPKDTLMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[0428] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

[0429] LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW

[0430] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0431] SEQ ID NO:104 IgG4 F405L / R409K

[0432] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS

[0433] GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV

[0434] FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY

[0435] RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK

[0436] NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEG

[0437] NVFSCSVMHEALHNHYTQKSLSLSLGK

[0438] Bispecific antibodies can also be produced using designs such as buttons (Genentech), CrossMAbs (Roche) and electrostatic matching (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono) and Biclonic (Merus).

[0439] In the "knob" strategy (see, for example, International Publication WO 2006 / 028936), selected amino acids that form the junction of the CH3 domains in human IgG can be mutated at positions that affect the interaction of the CH3 domains, thereby promoting heterodimer formation. Amino acids with small side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chain of an antibody that specifically binds to a second antigen. After the two antibodies are co-expressed, a heterodimer is formed due to the preferential interaction of the heavy chain with the "knob" with the heavy chain with the "knob". Exemplary CH3 substitution pairs that form knobs and buckles (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0440] In addition to utilizing a "buttons" strategy to facilitate Fab wall exchange, CrossMAb technology also utilizes CH1 / CL domain exchange in one half-arm to ensure correct light chain pairing of the resulting bispecific antibody (see, e.g., U.S. Patent 8,242,247).

[0441] Other exchange strategies can be used to generate full-length bispecific antibodies of the invention as follows: in one or both arms of the bispecific antibody, variable domains or constant domains, or both domains, are exchanged between the heavy chain and the light chain or within the heavy chain. These exchanges include, for example, VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1, as described in International Patent Publications WO2009 / 080254, WO2009 / 080251, WO2009 / 018386, and WO2009 / 080252.

[0442] Other strategies may also be used, such as using electrostatic interactions to promote heavy chain heterodimerization by replacing positively charged residues on one CH3 surface and replacing negatively charged residues on the second CH3 surface, as described in US Patent Publication US2010 / 0015133; US Patent Publication US2009 / 0182127; US Patent Publication US2010 / 028637 or US Patent Publication US2011 / 0123532. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351 Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W, as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849.

[0443] SEEDbody technology can be used to generate bispecific antibodies of the invention. SEEDbodies have selected IgG residues substituted with IgA residues in their constant domains to promote heterodimerization, as described in US Pat. No. 20070287170.

[0444] Mutations are generally made using standard methods, from the DNA level to the molecular level, such as the constant domains of antibodies.

[0445] Polynucleotides, vectors and host cells

[0446] The present invention also provides isolated polynucleotides, which encode anti-TMEFF2 antibodies or antigen-binding fragments thereof of the present invention. The present invention also provides isolated polynucleotides, which encode bispecific anti-TMEFF2 / anti-CD3 antibodies or antigen-binding fragments thereof of the present invention. The isolated polynucleotides capable of encoding the variable domain segments provided herein may be included on the same or different vectors to produce antibodies or antigen-binding fragments. The polynucleotides may be complementary deoxynucleic acids (cDNA) and may be codon-optimized for expression in a suitable host. Codon optimization is a well-known technique.

[0447] In some embodiments, the polynucleotides described herein (and the peptides encoded therein) comprise a leader sequence. Any leader sequence known in the art may be employed. The leader sequence may comprise a restriction site or a translation initiation site.

[0448] The present invention also provides an isolated polynucleotide encoding the VH of the antibody of the present invention, the VL of the antibody of the present invention, the heavy chain of the antibody of the present invention, or the light chain of the antibody of the present invention.

[0449] The present invention also provides an isolated polynucleotide encoding the VH of SEQ ID NO: 25, 26, 27, 87 or 89.

[0450] The present invention also provides an isolated polynucleotide encoding the VL of SEQ ID NO: 28, 29, 30, 31, 88 or 90.

[0451] The present invention also provides an isolated polynucleotide encoding HC1, LC1, HC2 or LC2 of the anti-TMEFF2 / anti-CD3 antibody of the present invention.

[0452] The present invention also provides an isolated polynucleotide encoding HCl of SEQ ID NO: 32, 33, 34, 91 or 93.

[0453] The present invention also provides an isolated polynucleotide encoding LC1 of SEQ ID NO: 35, 36, 37, 38, 92 or 94.

[0454] The present invention also provides an isolated polynucleotide encoding HC2 of SEQ ID NO: 76 or 78.

[0455] The present invention also provides an isolated polynucleotide encoding LC2 of SEQ ID NO: 77 or 79.

[0456] The present invention also provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO:39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 95, 96, 97, 98, 99, 100, 101 or 102.

[0457] The present invention also provides an isolated polynucleotide comprising the polynucleotide sequences of SEQ ID NOs: 105, 106, 80, 81, 107, 108, 82 and 83.

[0458] The polynucleotide sequence encoding the VH or VL of the antibody of the present invention or its antigen-binding fragment, or the heavy chain and light chain of the antibody of the present invention can be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow the nucleotide sequence to be expressed in the intended host cell. The polynucleotide can be cDNA.

[0459] The present invention also provides a vector comprising a polynucleotide of the present invention. Such vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vectors suitable for introducing the synthetic polynucleotide of the present invention into a given organism or genetic background by any means. For example, a polynucleotide encoding a light chain variable region and / or a heavy chain variable region of an antibody of the present invention, which can be optionally connected to a constant region, is inserted into an expression vector. The light chain and / or the heavy chain can be cloned in the same or different expression vectors. The DNA fragment encoding the immunoglobulin chain can be operably connected to a control sequence in one or more expression vectors that ensures the expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated or heterologous promoters), enhancer elements, and transcription terminator sequences are selected to be compatible with the host cell selected for expressing the antibody. After the vector is bound to an appropriate host, the host is maintained under conditions suitable for high-level expression of the protein, which is encoded by the bound polynucleotide.

[0460] Recombinant expression vectors within the scope of this specification include synthetic or cDNA-derived nucleic acid fragments that encode at least one recombinant protein such as VH, VL, HC or LC of an antibody that is operably connected to a suitable regulatory element. Such regulatory elements may include a transcription promoter, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation. Expression vectors, particularly mammalian expression vectors, may also include one or more non-transcriptional elements, such as a replication origin, a suitable promoter and enhancer connected to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (such as necessary ribosome binding sites), polyadenylation sites, splicing donor and acceptor sites, or transcription termination sequences. A replication origin that confers replication capability in a host may also be incorporated.

[0461] Transcriptional and translational control sequences in expression vectors used to transform vertebrate cells may be provided by viral sources. Exemplary vectors are described in Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0462] In some embodiments, the antibody coding sequence or antigen binding fragment coding sequence is placed under the control of a strong constitutive promoter (such as the promoter for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin, human myosin, human hemoglobin, human muscle creatine, etc.). In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use with the embodiments described. Such viral promoters include, but are not limited to, the immediate early promoter of cytomegalovirus (CMV), the early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) of Maroney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In one embodiment, the anti-TMEFF2 antibody or antigen-binding fragment thereof coding sequence is placed under the control of an inducible promoter (such as a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, a promoter containing one or more interferon-stimulated response elements (ISREs) (such as protein kinase R 2',5'-oligoadenylate synthetase, Mx gene, ADAR1, etc.)).

[0463] The vectors described herein may contain one or more internal ribosome entry sites (IRES). The inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing the expression of some proteins. In some embodiments, the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the above-mentioned nucleic acid sequences. The vector components may be connected continuously, or arranged in a manner that provides the best spacing for expressing the gene product (i.e., by introducing a "spacer" nucleotide between the ORFs), or positioned in another manner. Regulatory elements such as the IRES motif may also be arranged to provide the best spacing for expression.

[0464] The vector may include a selection marker well known in the art. The selection marker includes positive and negative selection markers, for example, antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene), glutamine synthase gene, HSV-TK, HSV-TK derivatives for ganciclovir selection or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al., 7Gene Ther.1738-1743 (2000)). The nucleic acid sequence encoding the selection marker or cloning site can be upstream or downstream of the nucleic acid sequence encoding the polypeptide of interest or the cloning site.

[0465] Exemplary vectors that can be used are bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza).

[0466] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:25 and / or the VL of SEQ ID NO:28.

[0467] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:39 and / or the polynucleotide of SEQ ID NO:42.

[0468] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:26 and / or the VL of SEQ ID NO:29.

[0469] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:40 and / or the polynucleotide of SEQ ID NO:43.

[0470] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:27 and / or the VL of SEQ ID NO:30.

[0471] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:41 and / or the polynucleotide of SEQ ID NO:44.

[0472] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:26 and / or the VL of SEQ ID NO:31.

[0473] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:40 and / or the polynucleotide of SEQ ID NO:45.

[0474] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:87 and / or the VL of SEQ ID NO:88.

[0475] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:95 and / or the polynucleotide of SEQ ID NO:96.

[0476] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:89 and / or the VL of SEQ ID NO:90.

[0477] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:99 and / or the polynucleotide of SEQ ID NO:100.

[0478] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:66 and / or the VL of SEQ ID NO:67.

[0479] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:105 and / or the polynucleotide of SEQ ID NO:106.

[0480] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:74 and / or the VL of SEQ ID NO:75.

[0481] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:107 and / or the polynucleotide of SEQ ID NO:108.

[0482] In some embodiments, the vector comprises a polynucleotide encoding the HC of SEQ ID NO:32 and / or the LC of SEQ ID NO:35.

[0483] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:46 and / or the polynucleotide of SEQ ID NO:49.

[0484] In some embodiments, the vector comprises a polynucleotide encoding the HC of SEQ ID NO:33 and / or the LC of SEQ ID NO:36.

[0485] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:47 and / or the polynucleotide of SEQ ID NO:50.

[0486] In some embodiments, the vector comprises a polynucleotide encoding the HC of SEQ ID NO:34 and / or the LC of SEQ ID NO:37.

[0487] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:48 and / or the polynucleotide of SEQ ID NO:51.

[0488] In some embodiments, the vector comprises a polynucleotide encoding the HC of SEQ ID NO:33 and / or the LC of SEQ ID NO:38.

[0489] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:47 and / or the polynucleotide of SEQ ID NO:52.

[0490] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:91 and / or the VL of SEQ ID NO:92.

[0491] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:97 and / or the polynucleotide of SEQ ID NO:98.

[0492] In some embodiments, the vector comprises a polynucleotide encoding the VH of SEQ ID NO:93 and / or the VL of SEQ ID NO:94.

[0493] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:101 and / or the polynucleotide of SEQ ID NO:102.

[0494] In some embodiments, the vector comprises a polynucleotide encoding the HC of SEQ ID NO:76 and / or the LC of SEQ ID NO:77.

[0495] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:80 and / or the polynucleotide of SEQ ID NO:81.

[0496] In some embodiments, the vector comprises a polynucleotide encoding the HC of SEQ ID NO:78 and / or the LC of SEQ ID NO:79.

[0497] In some embodiments, the antibody comprises the polynucleotide of SEQ ID NO:82 and / or the polynucleotide of SEQ ID NO:83.

[0498] The vectors described herein can be used to transform various cells with genes encoding the antibody or Fab. For example, the vector can be used to generate anti-TMEFF2 antibodies or cells that produce Fab. Therefore, the present invention also provides a host cell comprising one or more vectors of the present invention.

[0499] The techniques for introducing foreign genes into cells are known and can be used to construct the recombinant cells of the present invention.

[0500] "Host cell" refers to a cell into which a vector has been introduced. It should be understood that the term host cell is intended to refer not only to a particular subject cell, but also to the progeny of such a cell, and also to a stable cell line produced by a particular subject cell. Because certain modifications may occur in the offspring due to mutation or due to environmental influences, such progeny may be different from the parent cell, but are still included in the scope of the term "host cell" as used herein.

[0501] Such host cells can be eukaryotic cells, prokaryotic cells, plant cells or archaeal cells. Examples of prokaryotic host cells are Escherichia coli, bacilli such as Bacillus subtilis and other enterobacteriaceae such as Salmonella, Serratia and various Pseudomonas species. Other microorganisms such as yeast can also be used for expression. Saccharomyces (e.g., Saccharomyces cerevisiae) and Pichia pastoris are examples of suitable yeast host cells. Exemplary eukaryotic cells can be mammals, insects, birds or other animal sources. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines, such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHOK1SV (Lonza Biologics, Walkersville, MD), CHOK2SV (Lonza), CHO-K1 (ATCC CRL-61) or DG44.

[0502] Cells transformed with expression vectors as described herein can be selected or screened for recombinant expression of antibodies or antigen-binding fragments as described herein. Recombinant positive cells are amplified and screened, and subclones that exhibit a desired phenotype (such as high-level expression, enhanced growth characteristics, or, for example, the ability to produce proteins with desired biochemical characteristics due to post-translational modifications of protein modification or changes) are screened. These phenotypes may be due to the inherent properties of a given subclone or due to mutations. Mutations can be affected by the use of chemicals, UV wavelength light, radiation, viruses, insertion mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

[0503] The present invention also provides a method for preparing an antibody of the present invention, the method comprising culturing a host cell of the present invention under conditions that express the antibody, and recovering the antibody produced by the host cell. Methods for preparing antibodies and purifying them are well known in the art. Once synthesized (chemically or recombinantly), all antibodies, dimers thereof, single light chains and / or heavy chains, or other antibody fragments such as VH and / or VI, can be purified according to standard procedures, including ammonium sulfate precipitation, affinity chromatography columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The subject antibody can be substantially pure, for example, at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% pure, or more pure, for example, free of contaminants, such as cell debris, macromolecules other than the subject antibody, etc.

[0504] Standard molecular biological methods can be used to incorporate the polynucleotide sequences of the present invention into vectors. Host cell transformation, culture, antibody expression and purification are accomplished using well-known methods.

[0505] The present invention also provides a method for preparing the anti-TMEFF2 antibody of the present invention, the method comprising:

[0506] introducing a first polynucleotide encoding the VH of the antibody and a second polynucleotide encoding the VL of the antibody into an expression vector;

[0507] transforming a host cell with the expression vector;

[0508] culturing the host cell in a culture medium under conditions such that VL and VH are expressed and antibodies are formed; and

[0509] The antibody is recovered from the host cells or culture medium.

[0510] Pharmaceutical composition / administration

[0511] The present invention also provides a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibody of the present invention can be prepared as a pharmaceutical composition containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient or vehicle with which the antibody of the present invention is administered. Such vehicles can be liquids, such as water and oils, including those derived from petroleum, animals, plants, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally do not contain particulate matter. They can be sterilized by conventional known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, stabilizers, thickeners, lubricants, and colorants, etc. The concentration of the antibodies of the invention in such pharmaceutical formulations may vary from less than about 0.5%, typically to at least about 1% to as much as 15% or 20% by weight, and may be selected according to the selected mode of administration, primarily based on desired dose, fluid volume, viscosity, etc. Suitable vehicles and formulations including other human proteins (e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092 (see especially pp. 958-989).

[0512] The mode of administration of the antibodies of the invention may be any suitable route, such as parenteral administration, for example intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, mucosal (oral, intranasal, intravaginal, rectal) or other means as known to those skilled in the art.

[0513] The antibodies of the invention may also be administered prophylactically to reduce the risk of developing a disease such as cancer.

[0514] Thus, the pharmaceutical composition of the present invention for intramuscular injection can be prepared to contain 1 mL of sterile buffered water and about 1 ng / kg to about 100 mg / kg, such as about 50 ng / kg to about 30 mg / kg or more preferably about 5 mg / kg to about 25 mg / kg of the antibody of the present invention.

[0515] Methods of using anti-TMEFF2 antibodies and bispecific anti-TMEFF2 / anti-CD3 antibodies

[0516] The present invention also provides a method of treating TMEFF2-positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof to treat the TMEFF2-positive cancer.

[0517] The present invention also provides a method of treating TMEFF2-positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-TMEFF2 antibody or an antigen-binding fragment thereof to treat the TMEFF2-positive cancer.

[0518] A variety of genes are associated with the development of prostate cancer. One of the proteins involved in the development and progression of prostate cancer and therefore an ideal target for new anti-prostate cancer therapies is a transmembrane protein with an EGF-like domain and two follistatin-like domains (TMEFF2). TMEFF2 is a type I transmembrane protein consisting of two follistatin-like (FS) domains, an EGF-like domain, a transmembrane (TM) domain, and a short cytoplasmic tail. The highest expression of TMEFF2 protein was detected in two organs: the brain and the prostate (Liang et al. 2000; Horie et al. 2000). Elevated expression of TMEFF2 is also present in prostate cancer cell lines and clinical samples (Glynne-Jones et al. 2001; Gery et al. 2002; Afar et al. 2004), suggesting that TMEFF2 plays an important role in the progression of prostate cancer. The expression of the Tmeff2 gene is under the control of the androgen receptor. A large number of androgen-dependent cancer patients show high levels of TMEFF2 mRNA.

[0519] Therefore, anti-TMEFF2 antibodies may become an important tool for diagnosis, prognosis or treatment of prostate cancer.

[0520] "Cancer" is intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. An exemplary TMEFF-positive cancer includes prostate cancer.

[0521] In some embodiments, the prostate cancer is adenocarcinoma.

[0522] In some embodiments, the prostate cancer is metastatic prostate cancer. In some embodiments, the prostate cancer has metastasized to the rectum, lymph nodes, or bones, or any combination thereof.

[0523] In some embodiments, the prostate cancer is relapsed or refractory prostate cancer.

[0524] In some embodiments, the prostate cancer is castration-resistant prostate cancer.

[0525] In some embodiments, the prostate cancer is sensitive to androgen deprivation therapy.

[0526] In some embodiments, the prostate cancer is insensitive to androgen deprivation therapy.

[0527] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof for use in therapy.

[0528] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof for use in treating TMEFF2-positive cancers.

[0529] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof for use in treating TMEFF2 prostate cancer.

[0530] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof for use in the manufacture of a medicament for treating TMEFF2-positive cancer.

[0531] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0532] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0533] The first domain comprises the VH of SEQ ID NO:25 and the VL of SEQ ID NO:28, and the second domain comprises the VH of SEQ ID NO:66 and the VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises a first heavy chain (HC1) of SEQ ID NO:32, a first light chain (LC1) of SEQ ID NO:35, a second heavy chain (HC2) of SEQ ID NO:76, and a second light chain (LC2) of SEQ ID NO:77.

[0534] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0535] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0536] The first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:32, LC1 of SEQ ID NO:35, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0537] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0538] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0539] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0540] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0541] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 19, 21 and 23, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0542] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:36, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0543] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0544] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0545] The first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0546] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0547] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 14, 17, 18, 20 and 24, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0548] The first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:34, LC1 of SEQ ID NO:37, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0549] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0550] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0551] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:76, and LC2 of SEQ ID NO:77.

[0552] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0553] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 11, 13, 16, 18, 20 and 22, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0554] The first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:33, LC1 of SEQ ID NO:38, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0555] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0556] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0557] The first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:91, LC1 of SEQ ID NO:92, HC2 of SEQ ID NO:76 and LC2 of SEQ ID NO:77.

[0558] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0559] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0560] The first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:91, LC1 of SEQ ID NO:92, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0561] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0562] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 60, 61, 62, 63, 64 and 65, respectively;

[0563] The first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:93, LC1 of SEQ ID NO:94, HC2 of SEQ ID NO:76 and LC2 of SEQ ID NO:77.

[0564] The present invention also provides an isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof for use in treating TMEFF2-positive cancers such as prostate cancer, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein

[0565] The first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively;

[0566] The first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75; and / or the bispecific anti-TMEFF2 / anti-CD3 antibody or its antigen-binding fragment comprises HC1 of SEQ ID NO:93, LC1 of SEQ ID NO:94, HC2 of SEQ ID NO:78 and LC2 of SEQ ID NO:79.

[0567] An antibody of the invention may be administered in combination with a second therapeutic agent.

[0568] In some embodiments, the second therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

[0569] Reagent test kit

[0570] The present invention also provides a kit comprising an anti-TMEFF2 antibody or a bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention. The kit can be used for therapeutic purposes and as a diagnostic kit. The kit can be used to detect the presence of TMEFF2, CD3, or TMEFF2 and CD3 in a sample.

[0571] In some embodiments, the kit comprises an antibody of the invention and a reagent for detecting the antibody. The kit may comprise one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or reagents that can be used to chelate or otherwise couple the antibody to a label or therapeutic agent, or a radiation protection composition; a device or other material for preparing the antibody for administration; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

[0572] In some embodiments, the kit comprises a container containing an antibody of the invention and instructions for use of the kit.

[0573] In some embodiments, the antibodies in the kit are labeled.

[0574] The present invention also provides a kit comprising an anti-TMEFF2 antibody, wherein the anti-TMEFF2 antibody comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28.

[0575] The present invention also provides a kit comprising an anti-TMEFF2 antibody, wherein the anti-TMEFF2 antibody comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29.

[0576] The present invention also provides a kit comprising an anti-TMEFF2 antibody, wherein the anti-TMEFF2 antibody comprises VH of SEQ ID NO: 27 and VL of SEQ ID NO: 30.

[0577] The present invention also provides a kit comprising an anti-TMEFF2 antibody, wherein the anti-TMEFF2 antibody comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31.

[0578] The present invention also provides a kit comprising an anti-TMEFF2 antibody, wherein the anti-TMEFF2 antibody comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88.

[0579] The present invention also provides a kit comprising an anti-TMEFF2 antibody, wherein the anti-TMEFF2 antibody comprises VH of SEQ ID NO: 89 and VL of SEQ ID NO: 90.

[0580] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67.

[0581] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:25 and VL of SEQ ID NO:28, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0582] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67.

[0583] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:29, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0584] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67.

[0585] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:27 and VL of SEQ ID NO:30, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0586] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67.

[0587] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:26 and VL of SEQ ID NO:31, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0588] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67.

[0589] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:87 and VL of SEQ ID NO:88, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0590] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:66 and VL of SEQ ID NO:67.

[0591] The present invention also provides a kit comprising a bispecific anti-TMEFF2 / anti-CD3 antibody, which comprises a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises VH of SEQ ID NO:89 and VL of SEQ ID NO:90, and the second domain comprises VH of SEQ ID NO:74 and VL of SEQ ID NO:75.

[0592] Methods for detecting TMEFF2 or CD3

[0593] The present invention also provides a method for detecting TMEFF2 in a sample, which comprises obtaining a sample, contacting the sample with the anti-TMEFF2 antibody of the present invention, and detecting the antibody bound to TMEFF2 in the sample.

[0594] The present invention also provides a method for detecting TMEFF2 and CD3 in a sample, the method comprising: obtaining a sample; contacting the sample with a bispecific anti-TMEFF2 / anti-CD3 antibody of the present invention comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3; and detecting the antibody that binds to TMEFF2 and CD3 in the sample.

[0595] In some embodiments, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy tissue sections, including fine needle aspirate tissue), histological preparations, etc.

[0596] The antibodies of the invention described herein that bind to TMEFF2 or TMEFF2 and CD3 can be detected using known methods. Exemplary methods include directly labeling antibodies using fluorescent or chemiluminescent labels, or radiolabels, or attaching easily detectable moieties (such as biotin, enzymes, or epitope tags) to the antibodies of the invention. Exemplary labels and moieties are ruthenium, 111In-DOTA, 111In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β-galactosidase, polyhistidine (HIS tags), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridinium dyes, rhodamine dyes, and dye.

[0597] The antibodies of the present invention can be used in a variety of assays to detect TMEFF2 or TMEFF2 and CD3 in a sample. Exemplary assays are Western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assays.

[0598] The invention will now be described with reference to the following specific, non-limiting examples.

[0599] Example 1: Antigen Generation

[0600] Human extracellular domain (ECD) TMEFF2 is produced based on UniProt accession number Q9UIK5 sequence.ECD construct is designed to have 6X His-tag and Avi-tag sequence (construct TMEW1 at C-terminus; SEQ ID NO:6).Construct (amino acid 151-320) containing FS2 and EGF domain is designed to have human serum albumin (HSA) fusion with 6X His-tag and Avi-tag sequence (construct TMEW7; SEQ ID NO:7).Construct containing TMEFF2 membrane proximal domain (residue 230-320) is designed to have 6xHis tag (construct TMEW19; SEQ ID NO:8) or fused to rat IgG1 Fc (construct TMEW20; SEQ ID NO:9) with His tag.Residue 230-320 of TMEFF2 contains EGF domain across residue 261-301 of TMEFF2. Human TMEFF2 ECD expression constructs were transiently transfected into HEK293 derivative cells Expi293 (Gibco / Thermo Fisher Scientific) using Expifectamine according to the manufacturer's protocol. Prior to harvesting, cells were incubated at 8% CO on an orbital shaker. 2 Incubate at 37°C for 5 days.Expressed cells were removed by centrifugation, and immobilized metal affinity chromatography was used, using Ni Sepharose 6Fast Flow resin (GE Healthcare), followed by Superdex 200 preparative size exclusion chromatography (SEC) (GE Healthcare) in the Dubelcco phosphate buffer (1X DPBS) of pH 7.2 to purify the soluble TMEFF2 protein with His tag from culture medium. The amino acid sequence of the antigen generated is shown in Table 3.

[0601] Table 3.

[0602]

[0603]

[0604] Example 2. Generation of anti-TMEFF2 antibodies

[0605] Using human immunoglobulin loci Transgenic rats to produce antibodies Contains a chimeric human / rat IgH locus (comprising 22 human V H , all people D and J H The fragment was linked to rat C H locus) together with the complete human IgL locus (12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ). (See, e.g., Osborn et al., 2013, J Immunol, Vol. 190, No. 4, pp. 1481-1490). As a result, the rats exhibit reduced expression of rat immunoglobulins, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity chimeric human / rat IgG monoclonal antibodies with fully human variable regions. WO14 / 093908 describes Preparation and use of rats and the genome modifications carried by these rats.

[0606] OmniRats were immunized with human TMEFF2 construct FS2-EGF-Tev-HSA (C34S) -His-Avitag (TMEW7, SEQ ID NO: 7) and enhanced with construct spTMEFF2 (230-320) G3S-rat IgG1Fc (TMEW20, SEQ ID NO: 9). After the 89-day immunization schedule, lymph nodes were harvested from rats and used to generate hybridomas, and hybridoma supernatants were screened for binding to human TMEFF2-FL-ECD-His-Avi tag (TMEW1) protein by ELISA and / or SPARCL (spatial proximity to analyte reagent capture luminescence). Several supernatants were selected for secondary ELISA and SPARCL screening to screen for binding to TMEFF ECD, FS2-EGF, or only the EGF domain TMEFF2. Based on the screening results, several hybridoma clones were sequenced, expressed, and functionally characterized.

[0607] Generation of antibodies from phage display libraries

[0608] TMEFF2 binding Fabs were selected from two sets of de novo pIX phage display libraries using standard methods as described in Shi et al., J Mol Biol 397:385-96, 2010 and WO2009 / 085462. Briefly, two sets of libraries (called V3.0 and V5.0) were generated by diversifying human scaffolds, in which the germline VH genes IGHV1-69*01, IGHV3-23*01, and IGHV5-51*01 were recombined with the human IGHJ-4 minigene via the H3 loop (the IGHJ-6 minigene was also used in V5.0), and the human germline VLκ genes O12 (IGKV1-39*01), L6 (IGKV3-11*01), A27 (IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1 minigene to assemble complete VH and VL domains. Positions in the heavy and light chain variable regions around the H1, H2, L1, L2, and L3 loops were selected for diversification, which are frequently in contact with protein and peptide antigens. The sequence diversity at the selected position is limited to the residues that appear at each position in the IGHV or IGLV germline gene family of each IGHV or IGLV gene. Diversity is produced at the H3 loop by using a short to medium-sized synthetic loop of 7-14 amino acids in length for the V3.0 library and a synthetic loop of 6-19 amino acids in length for the V5.0 library. The amino acid distribution at H3 is designed to mimic the amino acid variation observed in human antibodies. According to their human VH and VL germline gene origins, the scaffolds for generating the libraries are named. For V3.0 and V5.0 groups, each of the three heavy chain libraries is combined with four germline light chains or four germline light chain libraries to produce 12 unique VH:VL combinations for each group of libraries for selection experiments.

[0609] V region cloning

[0610] Total RNA from phage hybridoma cell lysates was purified using the RNeasy 96 kit (Qiagen) according to the manufacturer's protocol. The resulting RNA was quantified using Drop Sense and stored at -80°C or used for cDNA synthesis using the Invitrogen SuperScript III First Strand Synthesis System by RT-PCR (Invitrogen). First-strand cDNA synthesis was performed using gene-specific primers annealed to the constant regions of the heavy chain, kappa chain, and lambda chain, respectively. A maximum of 3 μg of purified RNA, gene-specific primers, dNTP mix, reaction buffer, 25 mM MgCl 2 , DTT, RNaseOUT TM (40U / μL, Invitrogen) and SuperScriptTM The RT-PCR reaction mixture consisting of III RT (200U / μL, Invitrogen catalog number 18080-051) was incubated at 50°C for 50 minutes and at 85°C for 5 minutes. The resulting single-stranded cDNA was stored at -20°C or used directly for PCR amplification. The PCR reaction was performed using Platinum Pfx polymerase (Invitrogen). Using optimized PCR conditions, the v region fragment was amplified by forward primers and reverse primers that annealed to the leader sequences and constant regions of the heavy chain, k chain and lambda chain, respectively. The resulting PCR fragment was sequenced, and the amino acid sequence of the recovered v region was codon optimized and cloned into a pUnder-based expression vector carrying an IgG4 constant region (IgG4PAA isotype) with S228P, F234A and L235A mutations.

[0611] Expi293 small-scale transfection and purification

[0612] Selected antibodies identified from immunization campaigns or phage display were cloned and denoted as IgG1 PAA and purified via 2 mL small scale. TM Cells (ThermoFisher Scientific) were cultured at 1.25 × 10 5 -2.25×10 5 The density of viable cells / mL was inoculated in Expi293 TM Expression medium and incubate at 37°C, 7% CO 2 The cells were cultured in 125 mL-2 L shake flasks. When the density reached the logarithmic growth phase, the cells were plated at 3 × 10 6 -5×10 6 The viable cells / mL were subcultured and the survival rate was 98%-99%.

[0613] On the day of transfection, the viable cell density and percentage of viability were determined. Following the manufacturer's transfection protocol (ThermoFisher Publication No. MAN0007814), 3 × 10 6 Cells were transfected at a density of 10 viable cells / mL. Cultures were harvested on day 6 after transfection by centrifugation at 850×G for 15 minutes before purification. Antibodies were purified from the clarified supernatant using mAb Select Sure resin (GE Healthcare) and dialyzed into PBS. Protein concentration was determined by A280 measurement of the filtrate using a DropSense instrument (Trinean).

[0614] Example 3. Characterization of anti-TMEFF2 antibodies

[0615] Anti-TMEFF2 antibodies bind TMEFF2 with high affinity

[0616] The binding of selected IgG4PAA anti-TMEFF2 antibodies to TMEFF2 ECD (TMEW1: TMEFF2-FL ECD-His-Avi tag) and / or membrane proximal region (TMEW19: spTMEFF2 (230-320) G3S-H6) was assessed using Proteon (TMEB674, TMEB675, TMEB 565, and TMEB570) or Biacore SPR (TMEB762 and TMEB757). The kinetic parameters for binding to the selected antibodies are shown in Table 4. It was found that the anti-TMEFF2 antibodies bound to both TMEFF2 ECD and TMEFF2 membrane proximal region with picomolar affinity.

[0617] Table 4.

[0618]

[0619] ProteOn SPR

[0620] Anti-TMEFF2 mAb was measured by ProteOn SPR (Bio-Rad) to bind to the membrane proximal region of ECD and human TMEFF2. Purified mAb (diluted to a final concentration of 1 μg / mL in PBST) was used as a ligand in the assay and was fixed to goat anti-human (GAH) IgG Fc by Fc capture. For the amino coupling of GAH IgG Fc, a 1:1 mixture of EDC (40mM) and NHS (10mM) was mixed immediately before injection to activate the chip surface and inject in a vertical orientation. Then the GAH-Fc (with 30 μg / mL) antibody in acetate buffer (pH 5.0) was flowed on the surface for 300 seconds at a flow rate of 30 μL / min in a vertical orientation. Subsequently, any remaining reactive carboxyl groups in the surface were inactivated by injecting 1M ethanolamine (pH 8.5) along the same orientation. Antibodies were used for fixation at a concentration of 1 μg / mL. Antibodies flowed on the surface in a horizontal direction. The human TMEFF2 ECD or membrane proximal region was flowed in a vertical orientation with a 3-fold dilution series of 5 concentrations (the highest concentration in the range of 100-600nM) as an analyte to bind to the captured molecules. The buffer sample was also injected into the 6th channel in a vertical direction to monitor any drift of the baseline signal. The association phase and dissociation phase of all concentrations were monitored at a flow rate of 100 μL / min within 3 minutes and 30 (or 15) minutes, respectively. The binding surface was regenerated for the next interaction cycle using an 18-second pulse of 0.8% phosphoric acid to remove the bound antigen. The raw data was processed by subtracting two sets of reference data from the response data: 1) the inter-point signal to correct the nonspecific interaction between the antigen and the empty chip surface; 2) the empty channel signal (where only PBST flows on the chip) to correct the nonspecific baseline drift.

[0621] Biacore 8K SPR

[0622] Anti-TMEFF2 mAb is measured by Biacore 8K SPR and is combined with human TMEFF2 ECD. The form of determination is to use high-density anti-human Fc surface to capture mAb, and then use single-cycle kinetic method to inject human TMEFF2 concentration titration. Goat anti-human Fc IgG (Jackson Immunoresearch, catalog number 109-005-098) is directly fixed on flow cell 1 and 2 with 30 μg / mL in 10mM acetate buffer (pH 4.5) via amino coupling, and is directly fixed on CM5 sensor chip (GE) with a flow rate of 30 μL / min in HBSP (GE) buffer. mAb is captured on anti-human Fc IgG surface with 0.5 μg / mL (about 200-300RU) on flow cell 2. Then the running buffer is changed to HBSP+100 μg / mL BSA. Using a single cycle kinetics method, a 3-fold dilution series of TMEFF2 ECD with a concentration of 30nM was injected from low concentration to high concentration. The dissociation rate was monitored 30 minutes after the last or highest concentration injection, and then the surface was regenerated using 0.8% phosphoric acid (Bio-Rad). The buffer blank run with the same mAb captured and the same sample running conditions was also completed. The raw data was processed by subtracting two sets of reference data from the response data: 1) subtracting the reference flow cell 1 from the sample flow cell 2, and 2) subtracting the buffer blank run from the experimental run. The processing data of each mAb at all concentrations was globally fitted to a 1:1 simple Langmuir binding model to extract the estimated values ​​of kinetics (kon, koff) and affinity (KD) constants.

[0623] Thermal stability of anti-TMEFF2 antibodies

[0624] It was found by DSC (differential scanning calorimetry) that TMEB675 showed lower than usual thermal stability characteristics, with Tm=52°C at the onset of unfolding and the first thermal transition (Tm1) of 60.4°C. A more detailed examination of the sequence of TMEB675 (see Example 4) showed that somatic hypermutations (SHMs) were present in the framework regions of the heavy and light chains. Multiple reengineered variants were subcloned, expressed, purified and analyzed by DSC. The resulting mAbs TMEB762 and TMEFB757 showed the desired thermal stability characteristics (Tm1=69.4°C and Tm1=69.7°C, respectively). Compared to TMEB675, TMEB762 has the following amino acid modifications in the heavy chain: R14P, P20L and H81Q, while TMEFB757 has the following amino acid modifications in the heavy chain: R14P and P20L. Compared to TMEB675, TMEB762 has the following amino acid modifications in the light chain: A1D and A91P, while TMEFB757 has an A91P modification in the light chain. Residues are numbered according to Kabat. The kinetic parameters for the binding of TMEB675, TMEB762 to TMEFF2 ECD are shown in Table 4.

[0625] Example 4. Structural Characterization of Anti-TMEFF2 Antibodies

[0626] The cDNA sequence and amino acid translation of the antibody are obtained using standard techniques. In polypeptide sequence determination, several antibody cDNAs encoding variable regions or full-length antibodies are codon-optimized for large-scale expression using standard methods.

[0627] Table 5 shows the HCDR1 and HCDR2 amino acid sequences of selected anti-TMEFF2 antibodies.

[0628] Table 6 shows the HCDR3 amino acid sequences of selected anti-TMEFF2 antibodies.

[0629] Table 7 shows the LCDR1 and LCDR2 amino acid sequences of selected anti-TMEFF2 antibodies.

[0630] Table 8 shows the LCDR3 amino acid sequences of selected anti-TMEFF2 antibodies.

[0631] Table 9 shows the VH and VL amino acid sequences of selected anti-TMEFF2 antibodies.

[0632] Table 10 shows the SEQ ID NOs: of the heavy and light chains of selected anti-TMEFF2 antibodies.

[0633] Table 11 shows the heavy chain amino acid sequences of selected anti-TMEFF2 antibodies.

[0634] Table 12 shows the light chain amino acid sequences of selected anti-TMEFF2 antibodies.

[0635] Table 13 shows the SEQ ID NOs of the polynucleotides encoding various anti-TMEFF2 antibody chains.

[0636] Table 5.

[0637]

[0638] Table 6.

[0639]

[0640] Table 7.

[0641]

[0642] Table 8.

[0643] mAbs LCDR3 amino acids LCDR3 SEQ ID NO: TMEB675 LQDYNYALT 22 TMEB570 QQYGHSPIT 23 TMEB674 LQDYNYSLT 24 TMEB565 LQDYNYALT 22 TMEB762 LQDYNYPLT 86 TMEB757 LQDYNYPLT 86

[0644] Table 9.

[0645]

[0646]

[0647] Table 10.

[0648]

[0649] Table 11.

[0650]

[0651]

[0652] Table 12.

[0653]

[0654] Table 13.

[0655]

[0656] SEQ ID NO:39 (TMEB675 VH cDNA)

[0657] GAGGTGCAGCTGCTGGAAAGCGGCGGAGGCCTGGTGCAGAGAGGAGGAAGCCTGAGACCCAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAGCTGGGTCAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGAGCGTGATTAGCGGCAGCGGCGGCTTCACCGATTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAATAGCAAGAACACCCTGTACCTGCACATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGATGCCCCTGAACAGCCCTCACGACTACTGGGGCCAGGGAACCCTGGTGACCGTGTCCAGC

[0658] SEQ ID NO:40 (TMEB570, TMEB565 VH cDNA)

[0659] CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCGGCACCTTCAGCTCCTATTACATTAGCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGTGGCATTATCCCAATCAGTGGGCGTGCTAATTATGCGCAGAAATTTCAGGGCCGCGTGACCATTACCGCTGATGAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGACGGCTACAGTAGTGGACGTAGCACAACATACGCATTTGACTATTGGGGCCAGGGCACCCTGGTGACCGTGTCGAGT

[0660] SEQ ID NO:41 (TMEB674 VH cDNA)

[0661] GAAGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAGCCTCCTGGCGGAAGCCTGAGACTGAGCTGCGCCGCTAGCGGCTTCACCTTCAGCAGCTACAGCATGAGCTGGGTGAGACAGGCTCCTGGCAAGGGCCTGGAGTGGGTGAGCGTGATCAGCGGCGGAGGCAGCTTTACCAGCTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAACAACACCCTGTACCTGCAGATGAGCAGCCTGAGGGCCGAGGACACCGCCTTCTACTACTGCGCCAGGATGCCCCTGAACAGCCCCCATGACTGCTGGGGACAGGGCACCCTGGTGACCGTGAGCAGC

[0662] SEQ ID NO:42 (TMEB675 VL cDNA)

[0663] GCCATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACGCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAG

[0664] SEQ ID NO:43 (TMEB570 VL cDNA)

[0665] GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTTCCACATACTACCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTACGGTGCCTCCTATCGCGCGACCGGCATTCCGGATCGCTTTAGCGGCAGCGGTTCCGGCACCGATTTTACCCTGACCATTAGCCGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTACGGTCACAGCCCGATTACTTTTGGCCAGGGCACCAAAGTGGAAATCAAA

[0666] SEQ ID NO:44 (TMEB674 VL cDNA)

[0667] GCCATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGGAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGCAGCGGAAGCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACAGCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAGG

[0668] SEQ ID NO:45 (TMEB565 VL cDNA)

[0669] GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTGCCACCTATTATCTTGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTACGGTGCATCCTCCCGTGCGACCGGCATTCCGGATCGCTTTAGCGGCAGCGGTTCCGGCACCGATTTTACCCTGACCATTAGCCGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTACGGCTATAACCCAATTACCTTTGGCCAGGGCACCAAAGTGGAAATCAAA

[0670] SEQ ID NO:46 (TMEB675 HC cDNA)

[0671]

[0672] SEQ ID NO:47 (TMEB570, TMEB565 HC cDNA)

[0673]

[0674] SEQ ID NO:48 (TMEB674 HC cDNA)

[0675]

[0676] SEQ ID NO:49 (TMEB675 LC cDNA)

[0677] GCCATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACGCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0678] SEQ ID NO:50 (TMEB570 LC cDNA)

[0679] GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTTCCACATACTACCTGGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTACGGTGCCTCCTATCGCGCGACCGGCATTCCGGATCGCTTTAGCGGCAGCGGTTCCGGCACCGATTTTACCCTGACCATTAGCCGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTACGGTCACAGCCCGATTACTTTTGGCCAGGGCACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0680] SEQ ID NO:51 (TMEB674 LC cDNA)

[0681] GCCATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGGAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGCAGCGGAAGCGGCACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACAGCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAGGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0682] SEQ ID NO:52(TMEB565 LC cDNA)

[0683] GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCGTTGCCACCTATTATCTTGCGTGGTATCAGCAGAAACCGGGCCAGGCGCCGCGCCTGCTGATTTACGGTGCATCCTCCCGTGCGACCGGCATTCCGGATCGCTTTAGCGGCAGCGGTTCCGGCACCGATTTTACCCTGACCATTAGCCGCCTGGAACCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTACGGCTATAACCCAATTACCTTTGGCCAGGGCACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0684] SEQ ID NO:95 (TMEB762 VH cDNA)

[0685] GAGGTGCAGCTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCCGGAGGAAGCCTGAGACTCAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAGCTGGGTCAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGAGCGTGATTAGCGGCAGCGGCGGCTTCACCGATTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAATAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGATGCCCCTGAACAGCCCTCACGACTACTGGGGCCAGGGAACCCTGGTGACCGTGTCCAGC

[0686] SEQ ID NO:96 (TMEB762 VL cDNA)

[0687] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACCCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAG

[0688] SEQ ID NO:97 (TMEB762 HC cDNA)

[0689]

[0690] SEQ ID NO:98 (TMEB762 LC cDNA)

[0691] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACCCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0692] SEQ ID NO:99 (TMEB757 VH cDNA)GAGGTGCAGCTGCTGGAAAGCGGCGGAGGCCTGGTGCAGCCCGGAGGAAGCCTGAGACTCAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACAGCATGAGCTGGGTCAGGCAGGCCCCTGGCAAAGGACTGGAGTGGGTGAGCGTGATTAGCGGCAGCGGCGGCTTCACCGATTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAATAGCAAGAACACCCTGTACCTGCACATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGATGCCCCTGAACAGCCCTCACGACTACTGGGGCCAGGGAACCCTGGTGACCGTGTCCAGC

[0693] SEQ ID NO:100(TMEB757 VL cDNA)

[0694] GCCATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACCCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAG

[0695] SEQ ID NO:101(TMEB757 HC cDNA)

[0696]

[0697] SEQ ID NO:102(TMEB757 LC cDNA)

[0698] GCCATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACCTGCAGGGCCAGCCAGGGCATCAGAAACGACCTGGGCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGAGTGCCTAGCAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCTGCAGGACTACAACTACCCCCTGACATTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

[0699] The framework for the selected anti-TMEFF2 antibody is shown in Table 14.

[0700] Table 14.

[0701]

[0702] SEQ ID NO:53 (VH3_3-23 framework)

[0703] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGQGTLVTVSS

[0704] SEQ ID NO:54 (VH1_1-69 framework)

[0705] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

[0706] SEQ ID NO:55 (VKI_L11 framework)

[0707] AIQMTQSPSSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LQDYNYP

[0708] SEQ ID NO:56 (VKIII_A27 framework)

[0709] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP

[0710] Example 5. Epitope mapping of anti-TMEFF2 antibodies

[0711] Epitope mapping of TMEB570 and TMEB675 was performed using H / D exchange.TMEW1 (SEQ ID NO: 6) was used as a source of TMEFF2 in these assays.

[0712] 10 μg of TMEW1 in 130 μL of control buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4) was denatured by adding 130 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5) and incubating the mixture at 10°C for 3 minutes. The mixture was then subjected to on-column pepsin / protease XIII digestion using an in-house packed pepsin / protease XIII (w / w, 1:1) column (2.1 mm×30 mm). The resulting peptides were analyzed using a UPLC-MS system consisting of a Waters Acquity UPLC coupled to a Q Exactive™ hybrid quadrupole-orbitrap mass spectrometer (Thermo). Peptides were separated from 2% to 34% solvent B (0.2% formic acid in acetonitrile) on a 50 mm×1 mm C8 column with a gradient elution of 16.5 minutes. Solvent A was 0.2% formic acid in water. The injection valve and the pepsin / protease XIII column and its associated connecting tubing were located inside a cooling box maintained at 10°C. The second switching valve, C8 column and its associated stainless steel connecting tubing were located inside another cooling circulation box maintained at -6°C. Peptide identification was performed by searching the MS / MS data against the TMEW1 sequence using Mascot. The mass tolerances for precursor and product ions were 7 ppm and 0.02 Da, respectively.

[0713] 10 μ L of TMEW1 (5 μ g) or 10 μ L of TMEW1 and TMEB570 or TMEB675 mixture (5 μ g: 15 μ g) and 120 μ L of deuterium oxide labeling buffer (50 mM sodium phosphate, 100 mM sodium chloride, pH 7.4) were incubated at 10 ° C for 0 seconds, 30 seconds, 360 seconds, 3600 seconds or 14400 seconds. Hydrogen / deuterium (H / D) exchange at each time point was repeated twice. Hydrogen / deuterium exchange was quenched by adding 130 μ L of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (final pH 2.5). Subsequently, the quenched sample was subjected to pepsin / protease XIII digestion and LC-MS analysis on the column as described above. Mass spectrum was recorded only in MS mode.

[0714] Raw MS data were processed using HDX WorkBench software to analyze H / D exchange MS data (Pascal et al., J. Am. Soc. Mass Spectrom. 2012, 23(9), 1512-1521). Deuterium levels were calculated using the average mass difference between deuterated peptides and their native form (t0). Approximately 97%-99% of the protein could be mapped to a specific peptide. The slope of the deuterium accumulation curve showed significant differences over time as the peptide exchange progressed.

[0715] TMEB570 epitope: TMEW1 shows a modest reduction in deuterium uptake at residues 235-249 (residues are numbered according to SEQ ID NO: 2), for example residues HGKCEHSINMQEPSC (SEQ ID NO: 57) within the membrane proximal region do not undergo H / D exchange upon binding to TMEB570.

[0716] TMEB675 epitope: TMEW1 showed modest reduction in deuterium uptake at residues 252-268 (residues numbered according to SEQ ID NO: 2), for example residues DAGYTGQHCEKKDYSVL (SEQ ID NO: 58) within the membrane proximal region did not undergo H / D exchange upon binding to TMEB675.

[0717] Thus, the TMEB570 TMEFF2 epitope residues encompass HGKCEHSINMQEPSC (SEQ ID NO: 57) and the TMEB675 epitope residues encompass DAGYTGQHCEKKDYSVL (SEQ ID NO: 58). Both antibodies bind to TMEFF2 within the membrane proximal region.

[0718] Example 6. Generation of anti-CD3 antibodies

[0719] The anti-CD3 antibody CD3B219 has been described in US9850310.

[0720] By making OmniRat (OMT TM ) to generate additional anti-CD3 antibodies. The resulting hybridoma supernatants were screened for human and macaque CD3 + The binding of T lymphocytes, the clones were isolated and sequenced, and in some cases further engineered. The anti-CD3 antibodies were cloned into various isotypes, including effector-silencing IgG4 with S228P, F234A and L235A mutations, or IgG1 with L234A, L235A, G237A, P238S, H268A, A330S and P331S mutations. The two anti-CD3 antibodies generated were named CD3B376 and CD3B450.

[0721] The in vitro binding affinity of CD3B376 and CD3B450 to human T cells was determined by flow cytometry after hybridization to the antigen-specific target arms. Initial studies on human T cells were performed to determine the saturation binding constant (KdT) of the anti-CD3 tracer molecules. A fixed concentration of tracer ([T]) was then used in a competitive binding assay with titrated concentrations of the test mAb. The IC of the test molecules 50 The concentration at which 50% inhibition is achieved was used to determine the binding affinity (K d ): Kd =IC50 / (1+([T] / K d The saturation binding constant (K) of the tracer: commercially available AlexaFluor488 SP34-2 anti-CD3 (BioScience #557705) (data not shown) was determined using five human donors. d T).

[0722] Determine the saturation binding constant (K) of the tracer d T)

[0723] Methods: Human Pan T cells were cryopreserved in a nitrogen tank until use. T cells were thawed, washed with PBS, resuspended in FACS staining buffer, counted (with viability noted), and plated at 0.5×10 6 Resuspend at 1 μL / 1×10 6 Far Red Live / Dead stain (Life Technologies, AKA Invitrogen #L34974) was added to each cell (50 μL of DMSO into the vial); and FcR blocker (Miltenyi Biotec #130-059-901) (1 mL of 1:20 dilution / 0.5×10 6 Cells were inoculated at 50,000 cells / well and washed. Increasing concentrations of AlexaFluor488 SP-34 anti-CD3 were added to T cells and kept at 4°C for 2 hours. Cells were washed to remove unbound antibody, fixed for 15 minutes, washed, and resuspended in FACS staining buffer containing 1 mM EDTA.

[0724] Binding was measured using an iQue Intellicyte flow cytometer. Cells were gated for T cell populations, then single cells, then live cells (FL4). The geometric mean of staining was determined for each well (FL1).

[0725] The collected mean fluorescence intensity values ​​were plotted as a function of antibody molecule concentration and analyzed in a single-site binding assay (total binding) using Prism software. The software calculated the corresponding K d Value, which describes the binding of antibody molecules to receptors (CD3 on human Pan T cells), which follows the law of mass action. The formula is as follows: Y = (B max ×X) / (K d +X); where: Bmax is the maximum binding; K d is the concentration of ligand required to achieve half-maximal binding.

[0726] Results: Derived K for each donor dThe saturation binding constant (K) for human T cells was derived. d T) was 5.6±1.0 nM (n=4) and was used in the above formula to determine K d Binding affinity.

[0727] Anti-CD3 by competition assay Binding affinity of mAbs

[0728] Competition binding studies were performed using CD3B376 and CD3B450. Human Pan T cells were used to determine the binding affinity of the mAbs. The tracer used was the commercially available AlexaFluor488 SP-34 anti-CD3 (BioScience #557705) and the saturation binding constant of the tracer was as described above.

[0729] T cells were cryopreserved in a nitrogen tank until use. T cells were thawed, washed with PBS, resuspended in FACS staining buffer, counted, viability noted, and plated at 0.5 × 10 6 Resuspend at 1 μL / 1×10 6 FarRed Live / Dead stain (Life Technologies, AKA Invitrogen #L34974) (50 μL of DMSO to a vial) was added to each cell; and FcR blocker (Miltenyi Biotec #130-059-901) (1 mL of a 1:20 dilution / 0.5×10^6 cells) was added to the cells for 10 minutes each. The cells were seeded at 50,000 cells / well and washed.

[0730] mAbs (and isotype controls) were serially diluted 1:2 from a starting concentration of 1000 μg / mL or 200 μg / mL (2X), and a fixed concentration of tracer (5 μg / mL; 2X) was mixed together to give a 1X concentration. Thus, the final (1X) concentration of the tracer was 2.5 μg / mL = 16.6 nM. This mixture was added to T cells and kept at 4°C for 2 hours. The cells were then washed to remove unbound antibody, fixed for 15 minutes, washed, and resuspended in FACS staining buffer containing 1 mM EDTA.

[0731] Binding was measured using an iQue Intellicyte flow cytometer. Cells were gated for T cell populations, then single cells, then live cells (FL4). The geometric mean of staining was determined for each well (FL1). The collected mean fluorescence intensity values ​​were plotted as a function of log antibody molecule concentration (converted to nM) and analyzed using Prism software in a sigmoidal dose response (variable slope) from which EC50 / IC50 values ​​(in nM) were derived. The binding affinity (K) was derived using the following formula d ): K d =IC50 / (1+([T] / K d T)). Where: K d is the affinity of the competitor (unlabeled molecule); IC50 of the test compound (in nM); [T] is the concentration of the tracer (16.6 nM); K d T is the K of the tracer determined by saturation binding d (5.6 nM for humans).

[0732] The CD3B376 binding site is tighter than that of CD3B450 in both the bivalent and monovalent forms.

[0733] Table 15.

[0734]

[0735] The CDR, VH and VL amino acid sequences of CD3B219, CD3B376 and CD3B450 are shown in Table 16.

[0736] Table 16.

[0737]

[0738]

[0739] SEQ ID NO:76 CD3B219 HC; (IgG4 S228P, F234A, L235A, F405L and R409K)

[0740] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0741] SEQ ID NO:77 CD3B219 LC QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAVETTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0742] SEQ ID NO:78 CD3B376 HC (IgG4 S228P, F234A, L235A, F405L and R409K)

[0743] QVQLQQSGPRLVRPSQTLSLTCAISGDSVFNNNAAWSWIRQSPSRGLEWLGRTYYRSKWLYDYAVSVKSRITVNPDTSRNQFTLQLNSVTPEDTALYYCARGYSSSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0744] SEQ ID NO:79 CD3B376 LC

[0745] QSALTQPASVSGSPGQSITISCTGTSSNIGTYKFVSWYQQHPDKAPKVLLYEVSKRPSGVSSRFSGSKSGNTASLTISGLQAEDQADYHCVSYAGSGTLLFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0746] Table 17 shows the SEQ ID NO: of the polynucleotide encoding the anti-CD3 antibody chain.

[0747] Table 17

[0748]

[0749] SEQ ID NO:105CD3B219 VH cDNA

[0750] GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGGCCCGGATCAGAAGCAAGTACAACAATTACGCCACCTACTACGCCGCCTCCGTGAAGGGCAGATTCACCATCAGCCGGGACGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACCGCCGTGTACTACTGCGCCAGACACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCT

[0751] SEQ ID NO:106 CD3B219 VL cDNA

[0752] CAGACCGTCGTGACCCAGGAACCTAGCCTGACCGTGTCTCCTGGCGGCACCGTGACCCTGACCTGCAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTG

[0753] SEQ ID NO:80 CD3B219 HC cDNA

[0754]

[0755] SEQ ID NO:81 CD3B219 LC cDNA

[0756] CAGACCGTCGTGACCCAGGAACCTAGCCTGACCGTGTCTCCTGGCGGCACCGTGACCCTGACCTGCAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCAGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGTGCAGCCTGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGGGTCAGCCCAAGGCTGCACCCAGTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCCGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0757] SEQ ID NO: 107 CD3B376 VH cDNA

[0758] CAGGTGCAGCTGCAGCAGTCTGGCCCTAGACTCGTGCGGCCTTCCCAGACCCTGTCTCTGACCTGTGCCATCTCCGGCGACTCCGTGTTCAACAACAACGCCGCCTGGTCCTGGATCCGGCAGAGCCCTTCTAGAGGCCTGGAATGGCTGGGCCGGACCTACTACCGGTCCAAGTGGCTGTACGACTACGCCGTGTCCGTGAAGTCCCGGATCACCGTGAACCCTGACACCTCCCGGAACCAGTTCACCCTGCAGCTGAACTCCGTGACCCCTGAGGACACCGCCCTGTACTACTGCGCCAGAGGCTACTCCTCCTCCTTCGACTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCT

[0759] SEQ ID NO:108 CD3B376 VL cDNA

[0760] AGTCTGCTCTGACCCAGCCTGCCTCCGTGTCTGGCTCTCCCGGCCAGTCCATCACCATCAGCTGTACCGGCACCTCCTCCAACATCGGCACCTACAAGTTCGTGTCCTGGTATCAGCAGCACCCCGACAAGGCCCCCAAAGTGCTGCTGTACGAGGTGTCCAAGCGGCCCTCTGGCGTGTCCTCCAGATTCTCCGGCTCCAAGTCTGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCTGAGGACCAGGCCGACTACCACTGTGTGTCCTACGCTGGCTCTGGCACCCTGCTGTTTGGCGGAGGCACCAAGCTGACCGTGCTG

[0761] SEQ ID NO:82 CD3B376 HC cDNA

[0762]

[0763] SEQ ID NO:83 CD3B376 LC cDNA

[0764] CAGTCTGCTCTGACCCAGCCTGCTCCGTGTCTGGCTCTCCCGGCCAGTCCATCACCATCAGCTGTACCGGCACCTCCTCCAACATCGGCACCTACAAGTTCGTGTCCTGGTATCAGCAGCACCCCGACAAGGCCCCCAAAGTGCTGCTGTACGAGGTGTCC AAGCGGCCCTCTGGCGTGTCCTCCAGATTCTCCGGCTCCAAGTCTGGCAACACCGCCTCCCTGACCATCAGCGGACTGCAGGCTGAGGACCAGGCCGACTACCACTGTGTGTCCTACGCTGCCTCTGGCACCCTGCTGTTTGGCGGAGGCACCAAGCTGACC GTGCTGGGTCAGCCCAGGCTGCACCCAGTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCCGATAGCAGCCCCGTCAAGGCGGGA GTGGAGACCACCACAACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0765] Example 7. Generation of bispecific TMEFF2xCD3 antibodies

[0766] Selected monospecific anti-TMEFF2 and anti-CD3 antibodies are represented as IgG4 / κ. In the anti-CD3 antibody, F405L and R409K substitutions (EU numbering) were performed, while the anti-TMEFF2 antibody had a wild-type IgG4. In addition to position 405 and 409 substitutions, the IgG4 mAb was engineered to have S228P, F234A and L235A substitutions.

[0767] The monospecific antibodies were expressed and purified using a protein A column (HiTrap MabSelect SuRe column) using standard methods. After elution, the pool was dialyzed into D-PBS (pH 7.2).

[0768] Bispecific TMEFF2xCD3 antibodies were generated by combining monospecific TMEFF2 mAb and monospecific CD3 mAb in an in vitro Fab arm exchange, as described in International Patent Publication WO2011 / 131746. Briefly, approximately 1-20 mg / mL of each antibody in PBS (pH 7-7.4) and 75 mM 2-mercaptoethanolamine (2-MEA) were mixed together in a molar ratio of 1:1 and incubated at 25-37°C for 2-6 hours, after which 2-MEA was removed by dialysis, diafiltration, tangential flow filtration and / or spin cell filtration using standard methods.

[0769] Following in vitro Fab arm exchange, the bispecific antibodies were further purified using hydrophobic interaction chromatography to minimize residual parental anti-TMEFF2 and anti-CD3 antibodies using standard methods.

[0770] Tables 18 and 19 show the bispecific antibodies generated.

[0771] Table 18.

[0772]

[0773] Table 19.

[0774]

[0775] Example 8. Bispecific TMEFF2 × Characterization of CD3 antibodies

[0776] Purity analysis by analytical ultracentrifugation (AUC)

[0777] Analytical ultracentrifugation (AUC) allows the size, shape, aggregation state and reversible interaction of macromolecules in solution to be determined. Sedimentation velocity (SV) is a kind of AUC technique, which allows the concentration gradient of macromolecules to move to the outer radius of the sample holder (cell) when the centrifuge rotates. This realizes the determination of sedimentation coefficient, which is a factor of the size and shape of the molecule, and is unique for each molecule. For this purpose, Beckman Optima AUC instrument is used. The sample is loaded into a centrifuge equipped with a 1.2cm Beckman centerpiece (rated at 50Krpm) and a quartz window. The centrifuge is assembled and twisted to 130lbs. The centrifuge is placed in an An-50 (8 holes) or An-60 (4 holes) rotor and placed in the AUC chamber. Before starting the run, the temperature of the AUC is balanced to 20.5°C using the rotor in the chamber and continues for at least one hour. For mAb samples, run according to the following settings: 40K rpm, scan count (250 scans), scan collection frequency (90 seconds), data resolution (10 μM), 280nm wavelength. Use direct boundary fitting software SEDANAL to analyze data. Measure the purity of bispecific antibody TMCB150 and its parent mAb. TMCB150 shows 97.1% monomer, 2.8% dimer monomer and no aggregation, as determined by AUC to meet the acceptable standards for transiently expressed research materials for further biophysical characterization. TMEB762 shows 95.5% monomer, 4.5% dimer and no aggregation, while CD3B376 shows 97.7% monomer and 2.2% dimer, and no aggregation. Aggregate levels of >5% of the minimum two-step purified molecules can have a significant impact on biological activity, solubility, stability and storage life.

[0778] Conformational stability of anti-TMEFF2 / CD3 bispecific antibodies determined by DSC

[0779] The thermal unfolding of TMCB150 was determined by DSC (differential scanning calorimetry), which showed Tm = 52.6° C. at the beginning of unfolding, the first thermal transition (Tm1) was 61.8° C., the second thermal transition (Tm2) was 67.6° C., and the third thermal transition (Tm3) was 75.5° C. Based on the thermal transition characteristics of the parent antibodies (anti-TMEFF2, TMEB762 and anti-CD3, CD3B376) as evaluated by previous DSC, the Tm1 of TMCB150 corresponds to the CD3B376 FAB unfolding, and the Tm3 of TMCB150 corresponds to the TMEB762 Fab unfolding transition.

[0780] Serum stability

[0781] Develop a serum stability assay to evaluate the properties of lead candidates that bind to human serum components nonspecifically or off-target. This can predict poor pharmacokinetic and biodistribution properties. Fluorescence-based chromatography was used to evaluate the binding and stability of TMCB150 in both buffer and human serum. Bispecific antibodies were labeled with Alexa Fluor 488 conjugates (Invitrogen kit, according to the manufacturer's instructions), incubated in Hepes buffer and human serum (Sigma, catalog number H4522) for 2 days at 4 ° C and 37 ° C, and then analyzed by SEC-HPLC using an Agilent HPLC system equipped with a fluorescence detector. The percentage of aggregates was calculated by the integration of the area under the curve for each peak. TMCB150 showed 2.4% aggregation in Hepes buffer at time zero, and 2.0% and 1.3% aggregation were shown at 4 ° C and 37 ° C, respectively, two days later. In human serum, TMCB150 showed 1.7% aggregation at time zero, and 1.1% aggregation was shown at 4 ° C and 37 ° C, two days later.

[0782] Nonspecific binding

[0783] Nonspecific binding of lead molecules to irrelevant surfaces was determined by biosensor technology (Biacore 8K). The antibodies were passed over SPR surfaces coated with irrelevant proteins. If the antibody showed significant binding to these irrelevant surfaces, it was predicted that the antibody had poor in vivo properties and presented manufacturing difficulties. Lead molecules were tested at a final concentration of 1 μM. Irrelevant surfaces included negatively charged proteins (soybean trypsin inhibitor), positively charged proteins (lysozyme and β-defensin), hydrophobic proteins (Rh-integrin a4b7), human IgG, and sticky proteins (Rh-CD19). Appropriate controls were used in this experiment. Lead molecules flowed on two surfaces, one surface was blank and the other surface had molecules directly immobilized. The response unit (RU) level was determined by subtracting the blank RU from the test surface RU. The RU of TMCB150 and the parent mAb are given in the table below. Response units ≥ 100 predict a high risk of nonspecific binding to charged / hydrophobic / IgG surfaces, which will create challenges during manufacturing and translate into poor PK properties. None of the antibodies showed nonspecific binding to irrelevant surfaces, predicting low to no risk for manufacturing and in vivo behavior.

[0784] Table 20.

[0785] STI β-Defensin-3 Human IgG Lysozyme rh-integrin a4b7 rh-CD19 TMCB150 1.6 6.4 7.5 4.2 0 3.4 TMEB762 0 4.1 8.4 4.6 0 3.3 CD3B376 19.5 3.1 0.4 0.2 0 0

[0786] High concentration stability

[0787] Many monoclonal antibodies are formulated at high concentrations (≥100 mg / mL) to reduce the injection volume, which is conducive to subcutaneous administration. In addition, all monoclonal antibodies are exposed to transient high concentrations (≥50 mg / mL) during the purification process. Therefore, high concentration stability is a key quality attribute of these molecules. Concentration was performed using a centrifugal ultrafiltration device with a 30 kDa MWCO membrane. 5.1 mg to 5.3 mg of each protein was initially diluted to the same starting concentration and centrifuged at 4000 × g for 15 minutes. At the end of each 15-minute centrifugation step, the concentrator was removed from the centrifuge and a visual estimate of the remaining sample volume was recorded. The concentration was measured by a SoloVPE instrument. The concentrated sample was incubated at 4 ° C and 40 ° C for 2 weeks, and aliquots were taken at intervals to check integrity by analyzing SEC. TMCB150 and parent mAbs (CD3B376 and TMEB762) were normally concentrated. The final concentration of TMCB150 was 99.4 mg / mL, and the final concentration of the parent mAbs was 52.2 mg / mL (TMEB762) and 52.6 mg / mL (CD3B376). At 4°C and 40°C, the concentration remained the same over 2 weeks, indicating that the molecule has good intrinsic properties without the possibility of aggregation or adsorption to eppendorf tubes. The % of material (A: aggregates; M: monomers; F: fragments) as measured by SEC peak integration is provided in the table below. At high concentrations, after 2 weeks of storage at 40°C, the molecule was intact with 4-5% aggregates and ≤0.3% fragments, which predicts a good shelf life.

[0788] Table 21.

[0789]

[0790] Affinity of anti-TMEFF2 / CD3 bispecific antibodies determined by kinetic exclusion assay (KinExA)

[0791] The solution equilibrium affinity K of the bispecific mAb TMCB150 and its bivalent TMEFF2 parent TMEB762 mAb to the human TMEFF2 extracellular domain (ECD) was measured using a KinExA 3200 instrument (Sapidyne Instruments, Inc.). D. Serial dilutions of human TMEFF2 extracellular domain (ECD) were prepared with constant concentrations of anti-TMEFF2 mAb in 10mM HEPES, 150mM NaCl, 0.05% surfactant P20, 0.1% BSA and 0.02% NaN3 at pH 7.4. The reaction mixture was incubated at RT until the binding interaction reached equilibrium. The duration of incubation was determined using KinExA software simulation. Beads were prepared by direct covalent immobilization of TMEFF2-ECD by amino coupling on pre-activated beads consisting of bisacrylamide / azlactone copolymers (Pierce Biotechnology, Inc.). After incubation, samples were run on a KinExA instrument to assess free antibodies in the mixture by passing the mixture through TMEFF2-modified beads and detecting the captured antibodies using fluorescently labeled secondary antibodies. Using KinExA Pro software, data were fitted with a 1:1 binding model.

[0792] Table 22.

[0793] mAb samples <![CDATA[K D pM]]> <![CDATA[95%CI,K D pM]]> TMCB150 63.6 55.8-71.9 TMEB762 46.1 38.0-55.1

[0794] Measurement of Binding Affinity of Anti-TMEFF2 / CD3 Bispecific Antibodies to N-Terminal CD3ε Peptide

[0795] Kinetic rate constants were measured by SPR using Biacore 8K (GE Healthcare) and anti-human Fc biosensor surfaces. Anti-human immunoglobulin antibodies were covalently coupled to the surface of a CM4 sensor chip (GE Healthcare). The antibodies of interest were captured on the anti-human immunoglobulin sensor chip and then incubated in a 4% PBS solution containing 0.05% surfactant P20 (Tween TM 20) and 100μg / mL BSA in HEPES buffered saline with different concentrations of N-terminal CD3ε peptides. 30μL 0.8% phosphoric acid was injected twice at a rate of 100μL / min to regenerate the surface. The reported data is the difference in SPR signals between the flow cell containing the captured antibody and the reference cell without the captured antibody. The additional instrumental contribution to the signal is removed by subtracting the data of the blank injection from the signal deducted from the reference. The data are analyzed by fitting the association phase and dissociation phase (global fit) at all concentrations using the Biaevaluation software (Biacore, Inc.). The data are reported as mean + 95% CI (confidence interval), which is calculated by the square root of the t value * standard deviation / number of parallel determinations of 95% CI.

[0796] Table 23.

[0797]

[0798] Example 9. Bispecific TMEFF2xCD3 Antibody is Effective in T-Cell Mediated Killing of Prostate Cancer Cells of .

[0799] In vitro T cell-mediated killing of prostate cancer cells .

[0800] The ability of selected bispecific TMEFF2xCD3 antibodies to mediate T-cell mediated killing of prostate cancer cells was evaluated.

[0801] The killing of T-cell-mediated TMEFF2xCD3 bispecific antibodies was measured using a caspase cytotoxicity assay, which indirectly measures cell killing by cleaving a fluorescent substrate with active caspase 3 / 7. The cleavage of the substrate produces a fluorescent DNA dye, wherein the fluorescence is confined to the nucleus. Throughout the assay, a 10X mechanical objective capable of accurately imaging the wells at the same coordinates was used to perform repeated fluorescence measurements in each well. Based on defined size restrictions and / or by using a second marker to identify target cell populations.

[0802] Isolation of frozen Pan CD3 from normal healthy donors by negative selection + T cells (Biological Specialty Corporation, Colmar, PA). Prostate cancer cells (LNCaP-AR) expressing TMEFF2 were cultured in RPMI 1640 (Life Technologies) with 10% HI FBS + supplements. In the absence of a selection agent, T cells and target cells were combined in phenol red-free RPMI + 10% FBS and supplements (Life Technologies) at an effector to target ratio (E: T) of 3: 1, and 0.6 μL NucView caspase reagent (Essen Bioscience) was added to each mL of cells according to the manufacturer's instructions. Cells with a total volume of 0.1 mL were added to the appropriate wells of a transparent 96-well flat-bottom plate (BD Falcon). TMEFF2xCD3 bispecific antibodies were prepared at 2X final concentrations in phenol red-free RPMI prepared as shown above, and 0.1 mL of the compound was added to each well. After incubation at room temperature for 30 minutes to minimize cell aggregation at the edge of the well, the plate was transferred to a 37°C, 5% CO 2 The results were in a Zoom Incucyte instrument (Essen Bioscience).

[0803] Processing definitions on the Incucyte were designed according to the manufacturing guidelines for each cell line tested. Measurements were taken every six hours until a plateau in the caspase signal was observed, followed by three or more consecutive decreases from the maximum signal in the well containing the highest concentration of the test compound.

[0804] After completing the assay, each plate was analyzed using appropriate treatment definitions. Raw fluorescence data was exported from Incucyte Zoom software and pasted into GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Caspase 3 / 7 activity was determined by calculating the area under the curve (AUC) of each well in GraphPad. AUC values ​​were plotted as a function of Log10 nM compound. The EC50 of each dose curve in nanomolar (nM) was reported after nonlinear regression analysis (4 parameter fit, least squares method). Each assay contained a minimum of three biological parallel assay samples, and each cell line was tested using T cells from five healthy donors. Data were further analyzed using nonlinear regression models by nonclinical statistics.

[0805] Figure 3 Shown is the killing of LNCaP cells by selected bispecific TMEFF2xCD3 antibodies over time as measured by increase in caspase 3 / 7 activity.

[0806] The bispecific TMEFF2xCD3 antibody is effective in an in vivo prostate cancer tumor model

[0807] Selected bispecific antibodies were tested in an ex vivo LnCaP prostate cancer model in male NSG mice. For this study, 10 40 μg of 1 ... 6 A 50% Cultrex solution of LnCaP cells was administered to the right flank. When the tumor reached approximately 100-150 mm 3 The animals were randomized and injected intraperitoneally with 20 6 T cells / mouse. Ten animals per group. Antibody treatment started 1-3 days after T cell injection. Antibody was administered intraperitoneally twice a week. All animals received IVIG + Fc blocker prior to dosing. Tumor volume and body weight were assessed twice a week until tumors reached approximately 1200 mm 3 Treatment groups are shown in Table 24. CD3xNull antibody, which has a CD3B219 CD3 binding arm and an empty arm that binds HIVgpl20, was used as a negative control in these assays.

[0808] Table 24.

[0809] Group deal with dose 1 CD3xNull 5mg / kg 2 TMCB93 (TMEB675×CD3B219) 5mg / kg 3 TMCB93 (TMEB675×CD3B219) 0.5mg / kg 4 TMCB101(TMEB570×CD3B219) 5mg / kg 5 TMCB101(TMEB570×CD3B219) 0.5mg / kg 6 TMCB131(TMEB570×CD3B376) 5mg / kg 7 TMCB131(TMEB570×CD3B376) 0.5mg / kg 8 TMCB132(TMEB675×CD3B376) 5mg / kg 9 TMCB132(TMEB675×CD3B376) 0.5mg / kg

[0810] Table 25 shows the percentage of tumor growth inhibition in each group on day 38 after tumor transplantation.

[0811] Figure 4Shown is a decrease in mean tumor volume over time after tumor transplantation. Figure 5A Shown is the reduction in mean tumor volume per mouse treated with 0.5 mg / kg TMCB93. Figure 5B Shown is the reduction in mean tumor volume per mouse treated with 0.5 mg / kg TMCB132.

[0812] Table 25.

[0813] deal with TGI% at Day 38 CNTO7008(Null×CD3) TMCB93(TMEB675×CD3B219)5mg / kg 83.9 TMCB93(TMEB675×CD3B219)0.5mg / kg 75.6 TMCB132(TMEB675×CD3B37)5mg / kg 48 TMCB132(TMEB675×CD3B376)0.5mg / kg 47.7 TMCB101(TMEB570×CD3B219)5mg / kg 25 TMCB101(TMEB570×CD3B219)0.5mg / kg 38.3 TMCB131(TMEB570×CD3B376)5mg / kg 23.3 TMCB131(TMEB570×CD3B376)0.5mg / kg 35

[0814] In male NOD.Cg-Prkdc humanized with 20e6 T cells scid Iq tm1Wjl The efficacy of TMEB762xCD3B376 (TMCB150) was evaluated in established LNCaP xenografts in / SzJ (NSG) mice. At day 13 after tumor implantation, animals were randomized into groups of 10 animals by mean tumor volume. 0.5, 0.1 and 0.05 mg / kg of TMEB762xCD3B376 or 0.5 mg / kg of nullxCD3B376 antibody control were administered IP twice a week for 5 weeks. At day 35 after tumor implantation, when at least eight animals were retained in each group, tumor growth inhibition (%TGI) determined by tumor volume was calculated. Compared with the nullxCD3 control ( Figure 6 ) compared with the nullxCD3 treated control, statistically significant tumor growth inhibition was observed with 0.5mg / kg and 0.1mg / kg, but not 0.05mg / kg of TMEB762xCD3B376. Compared with the nullxCD3 treated control, 0.5mg / kg, 0.1mg / kg and 0.05mg / kg of TMEB762xCDB376 induced 110%, 88% and 25% tumor growth inhibition, respectively. Treatment with 0.5mg / kg and 0.1mg / kg of TMEB762xCDB376 resulted in seven and three complete responses, respectively. Subcutaneous LNCaP tumors were measured by caliper twice a week and the results are expressed as mean tumor volume (mm 3 ) ± SEM (≥8 mice remaining in each group).

[0815] T cell activation in response to administration of TMCB132

[0816] T cell activation in LnCaP prostate cancer cells was measured by flow cytometry, specifically by assessing CD25 positivity in CD3+ / CD8+ T cells from 6 individual normal healthy donors (9642, 9672, 9762, 9772, 9832, 9852) 72 hours after treatment with TMCB132 ( Figure 7). In response to administration of TMCB132 on LnCaP prostate cancer cells, activation of CD3+ pan-T cells added at a 3:1 effector:target ratio was observed.

[0817] T cell-mediated cytotoxicity of TMCB132 in vitro .

[0818] The potential cytotoxicity of TMCB132 in vitro was assessed using a T cell-mediated cytotoxicity assay using real-time time lapse imaging on the Incucyte platform. TMCB132 was tested in the TMEFF2-positive cell line LnCaP at a 3:1 (effector: target) effector: target ratio (E: T ratio) in the presence of isolated Pan human CD3+ T cells from healthy donors. Cell death caused by apoptosis was monitored by measuring the fluorescence signal of active caspase-3 / 7 over a period of 96 hours. TMCB132 promoted a dose-dependent decrease in viable LnCaP cells over time. Dose-dependent enhancement of caspase-3 / 7 activity or fluorescence signal indicated cell death in LnCaP cells in the presence of T cells ( Figure 8 ). The data indicate that TMCB132 is effective in inducing T cell-mediated death in LnCaP tumor cells.

[0819] Efficacy of TMCB132 in SC human prostate xenografts established in T-cell humanized mice .

[0820] The antitumor efficacy of TMCB132 was evaluated in established subcutaneous (SC) human prostate LNCaP xenografts in male NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG, The Jackson Laboratory, Bar Harbor, ME) mice humanized with 20e6 T cells. NSG male. On day 13 after tumor implantation, animals were randomized into groups of 10 animals by mean tumor volume. 0.5 mg / kg, 0.1 mg / kg and 0.05 mg / kg of TMCB132 or 0.5 mg / kg of nullxCD3B219 antibody control were administered IP twice a week for 4 weeks. On day 45 after tumor implantation, when at least seven animals were retained in each group, tumor growth inhibition (%TGI) determined by tumor volume was calculated. Compared with nullxCD3 control ( Fig. 9Statistically significant tumor growth inhibition was observed with 0.5 mg / kg, 0.1 mg / kg and 0.05 mg / kg of TMCB132 compared to nullxCD3 treated controls (p≤0.0001). TMCB132 at 0.5 mg / kg, 0.1 mg / kg and 0.05 mg / kg elicited 102%, 109% and 47% tumor growth inhibition, respectively. Treatment with 0.5 mg / kg, 0.1 mg / kg and 0.05 mg / kg of TMCB132 resulted in three, two and one complete responses, respectively.

Claims

1. An isolated bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof comprising a first domain that binds to TMEFF2 and a second domain that binds to CD3, wherein the first domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 10, 12, 15, 18, 20 and 86, respectively, and the second domain comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 68, 69, 70, 71, 72 and 73, respectively.

2. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1, wherein the antibody binds to the membrane proximal region of SEQ ID NO: 110 of TMEFF2.

3. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody competes with a reference antibody for binding to the membrane proximal region of TMEFF2, the reference antibody comprising: a) the heavy chain variable region (VH) of SEQ ID NO: 25 and the light chain variable region (VL) of SEQ ID NO: 28; or b) the VH of SEQ ID NO:26 and the VL of SEQ ID NO:29; c) the VH of SEQ ID NO:27 and the VL of SEQ ID NO:30; d) the VH of SEQ ID NO:26 and the VL of SEQ ID NO:31; e) the VH of SEQ ID NO: 87 and the VL of SEQ ID NO: 88; or f) the VH of SEQ ID NO:89 and the VL of SEQ ID NO:

90.

4. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody binds to the membrane proximal region of TMEFF2 within residue or DAGYTGQHCEKKDYSVL (SEQ ID NO: 58).

5. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is expressed at 0.4×10 -9 M or less dissociation constant (K D ) binds to the membrane proximal region of TMEFF2, wherein the K D Measurements were performed using surface plasmon resonance in acetate buffer at pH 4.5-5.0 at room temperature.

6. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 5, wherein the antibody is expressed at a concentration between 0.1×10 -10 M and 0.4×10 -9 The K between M D Binds to the membrane-proximal region of human TMEFF2.

7. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the first domain comprises the VH of SEQ ID NO: 87 and the VL of SEQ ID NO:

88.

8. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the second domain comprises the VH of SEQ ID NO: 74 and the VL of SEQ ID NO:

75.

9. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof according to claim 8, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprises HC1 of SEQ ID NO:91, LC1 of SEQ ID NO:92, HC2 of SEQ ID NO:78, and LC2 of SEQ ID NO:

79.

10. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is of IgG1, IgG2, IgG3 or IgG4 isotype.

11. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody comprises an alanine at position 234, an alanine at position 235, or an alanine at position 234 and an alanine at position 235 in the HC1, in the HC2, or in the HC1 and the HC2, wherein the residues are numbered according to the EU index.

12. The isolated bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises a proline at position 228 in the HC1 and in the HC2, wherein residues are numbered according to the EU index.

13. The isolated bispecific anti-TMEFF2 x CD3 antibody or the antigen-binding fragment thereof of claim 1 or 2, wherein the antibody comprises a phenylalanine at position 405 and an arginine at position 409 in the HC1, and comprises a leucine at position 405 and a lysine at position 409 in the HC2. 14 . A pharmaceutical composition comprising the bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier. 15 . A polynucleotide encoding the bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof according to claim 9 . A vector comprising the polynucleotide according to claim 15 .

17. A host cell comprising the vector according to claim 16.

18. A method for preparing a bispecific anti-TMEFF2 / anti-CD3 antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell according to claim 17 under conditions where the antibody is expressed, and recovering and purifying the bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof produced by the host cell.

19. A method for preparing the bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to claim 9, the method comprising: a) mixing a monospecific bivalent TMEFF2 antibody having two identical HC1s and two identical LC1s with a monospecific bivalent CD3 antibody having two identical HC2s and two identical LC2s in a 1:1 molar ratio; b) introducing a reducing agent into the mixture; c) incubating the mixture for ninety minutes to six hours; d) removing the reducing agent; and e) purifying the bispecific anti-TMEFF2 / anti-CD3 antibody or antigen-binding fragment thereof comprising the HC1, the LC1, the HC2 and the LC2.

20. The method of claim 19, wherein the reducing agent is 2-mercaptoethanolamine (2-MEA).

21. The method of claim 20, wherein the 2-MEA is present at a concentration of 25 mM to 75 mM.

22. The method according to any one of claims 19 to 21, wherein the incubation step is performed at a temperature of 25°C to 37°C.

23. Use of a therapeutically effective amount of the bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof according to any one of claims 9 to 13 or the pharmaceutical composition according to claim 14 in the preparation of a medicament for treating TMEFF2-positive cancer, wherein the TMEFF2-positive cancer is prostate cancer.

24. The use according to claim 23, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

25. The use according to any one of claims 22 to 24, wherein the bispecific anti-TMEFF2 / anti-CD3 antibody or the antigen-binding fragment thereof is administered in combination with a second therapeutic agent.

26. The use according to claim 25, wherein the second therapeutic agent is surgery, chemotherapy, androgen deprivation therapy or radiotherapy, or any combination thereof.

27. A kit comprising the antibody according to claim 9.

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