Anti-CD3 antibody folate bioconjugates and their uses

Abstract

To provide novel anti-CD3 folate antibodies.SOLUTION: An anti-CD3 antibody or antibody fragment is provided, which has at least one amino acid sequence among 62 specific amino acid sequences with mutually different sequences. The anti-CD3 antibody may incorporate one or more non-naturally encoded amino acids, and may further include one or more folate molecules. The non-naturally encoded amino acid may have a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azido group, or an alkyne group. The non-naturally encoded amino acid may be conjugated with a folate molecule.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Cross - reference to Related Applications] This application claims the benefit of U.S. Provisional Application No. 62 / 732,793, entitled "Anti - CD3 Fab Folates Antibodies and Their Uses," filed on August 28, 2018, the content of which is hereby incorporated by reference in its entirety.

[0002] [Sequence Listing] This application is filed via EFS - Web in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety. The name of the ASCII copy created on August 28, 2019, is AMBX_0228_00PCT_Sequence_Listing.txt, and the size is 128,417 bytes.

[0003] [Field of the Invention] The disclosure of the present invention relates to the field of immuno - oncology. More particularly, the present invention relates to anti - CD3 antibodies and fragments or variants thereof complexed with one or more folic acid molecules. The present invention also relates to anti - CD3 Fab - folate antibodies and variants thereof complexed with polyethylene glycol (PEG).

[0004] [Background of the Invention] Ovarian cancer is one of the most common cancers in women worldwide. Approximately 250,000 women are diagnosed with ovarian cancer each year, and approximately 140,000 women die from this disease each year. The five - year survival rate of ovarian cancer varies depending on the type and stage of the cancer, and generally, the more advanced the ovarian cancer, the worse the five - year survival rate. Current treatment options for ovarian cancer include chemotherapy, surgery, radiation, or a combination of these therapies. The response rate for advanced ovarian cancer after platinum - based chemotherapy following debulking surgery is 80%, and 40 - 60% have a complete response. Unfortunately, approximately 70% of these patients relapse, and the median progression - free survival period is 18 months.

[0005] Currently, there are drawbacks in the field of therapies targeting ovarian cancer, including ovarian cancer types such as ovarian cancer, and epithelial tumors, stromal tumors, and germ cell tumors, as well as fallopian tube cancer and primary peritoneal carcinomas. Surgery, radiation, and various chemotherapeutic agents are commonly used in the treatment of ovarian cancer patients. Recently, the FDA (U.S. Food and Drug Administration) has accepted an additional biological license application (sBLA) for bevacizumab (Avastin) for the treatment of advanced ovarian cancer as a primary treatment.

[0006] Immunotherapy is being evaluated as a new treatment option for cancer patients. In immunotherapy, biological agents or engineered T cells are used to stimulate the patient's immune system to address the patient's cancer. Some immunotherapies are very promising and have been approved for the treatment of various types of cancer. Examples of immunotherapeutic agents used in cancer patients include immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T), bispecific T cell engagers (BiTE), T cell-dependent bispecific antibodies (TDB) of various designs, NK cell-dependent bispecific antibodies, macrophage-dependent bispecific antibodies, autologous antigen-presenting cell (APC) / cancer vaccines.

[0007] Biological agents currently undergoing clinical trials for ovarian cancer patients include antibody-drug conjugates against folate receptor alpha (mirvetuximab soravtansine (IMGN853)), EpCAM-CD3 bispecific antibody (catumaxomab), DLL4-VEGF bispecific vaccine targeting NY-ESO-1, and CAR-T targeting folate receptor alpha (FOLR1), among others.

[0008] Ovarian cancer has been reported to have an immunosuppressive environment. This poses a challenge to the development of certain types of immunotherapeutic agents such as checkpoint inhibitors. Currently, immunotherapeutic agents for treating ovarian cancer patients are not approved, but some are being evaluated in clinical trials.

[0009] Folate receptors are expressed in many cancers, including but not limited to epithelial cancers such as breast cancer, cervical cancer, colorectal cancer, renal cancer, nasopharyngeal cancer, ovarian cancer, and endometrial cancer. The folate receptor (FR) family in humans includes FRα, FRβ, and FRγ. Folate receptor alpha (FOLR1) is a GPI-anchored receptor and is highly expressed in approximately 80-90% of ovarian cancers. FOLR1 binds folic acid, also known as vitamin B9, and 5-methyl-tetrahydrofolate (5-MTHF), the major metabolite of folic acid. High expression of FOLR1 is observed in approximately 76% of high-grade serous ovarian cancer, the main histological type, compared to 11% in mucinous ovarian cancer. FOLR1 has low and restricted expression in normal tissues, making it a preferred target for oncology drug development.

[0010] To overcome the drawbacks in the art, the inventors developed an antibody conjugate in an anti-CD3 antibody that incorporates one or more non-naturally encoded amino acids and further contains one or more folate molecules. The bispecific antibody can comprise or consist of an anti-CD3 antibody engineered to have one or more non-naturally encoded amino acids in the heavy or light chain of the Fab. The bispecific antibody or antibody fragment can include an anti-CD3 antibody engineered to have one or more non-naturally encoded amino acids in the heavy and / or light chain.

[0011] 〔Summary of the Invention〕 Anti-CD3 bispecific antibodies that accumulate cytotoxic T cells in cancer cells represent a promising new approach in the treatment of various liquid and solid tumors. The present invention provides anti-CD3 antibodies. In some embodiments, the anti-CD3 antibody is a bispecific antibody. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab. In some embodiments, the anti-CD3 antibody comprises amino acids that are not naturally encoded in the heavy or light chain of the Fab, preferably in the heavy chain. In some embodiments, the amino acids that are not naturally encoded are para-acetylphenylalanine (pAF). In some embodiments, the anti-CD3 antibody comprises two or more amino acids that are not naturally encoded in the heavy and / or light chain of the Fab, for example two, three, or four amino acids that are not naturally encoded, and optionally, the two or more amino acids that are not naturally encoded are pAF. In some embodiments, the anti-CD3 Fab is complexed with folate. In some embodiments, the anti-CD3 Fab is complexed with a water-soluble polymer (such as polyethylene glycol (PEG)). In some embodiments, the anti-CD3 Fab is complexed with both folate and polyethylene glycol (PEG), and optionally, complexed using a bifunctional linker. In some embodiments, the anti-CD3 Fab is complexed with two folate and two polyethylene glycol (PEG) molecules. In some embodiments, the complexation is carried out via the side chains of amino acids that are not naturally encoded (such as pAF). In some embodiments, the anti-CD3 antibody accumulates cytotoxic T cells in folate receptor positive (FR+) tumor cells. In some embodiments, the anti-CD3 antibody has improved efficacy, reduced toxicity, improved pharmacokinetic (PK) properties, improved affinity, improved tumor-associated antigen (TAA) binding, improved in vivo half-life (T1 / 2), improved in vitro activity, improved serum half-life, and / or improved in vivo activity. In some embodiments, the anti-CD3 antibody has improved efficacy. In some embodiments, the anti-CD3 antibody has reduced toxicity. In some embodiments, the anti-CD3 antibody has improved PK properties. In some embodiments, the anti-CD3 antibody has improved affinity.In some embodiments, the anti-CD3 antibody has improved TAA binding. In some embodiments, the anti-CD3 antibody has improved in vivo T1 / 2. In some embodiments, the anti-CD3 antibody has improved in vitro activity. In some embodiments, the anti-CD3 antibody has improved serum half-life. In some embodiments, the anti-CD3 antibody has improved in vivo activity. The present invention provides an optimization of an anti-CD3 Fab-folate conjugate. In this optimization, cytotoxic T cells are directed to folate receptor positive (FR+) tumor cells to obtain optimal efficacy, reduced toxicity, and optimal pharmacokinetic (PK) properties. For example, the present invention provides an optimized anti-CD3 Fab-folate conjugate. The optimized anti-CD3 Fab-folate conjugate directs cytotoxic T cells to folate receptor positive (FR+) tumor cells to obtain optimal efficacy, reduced toxicity, and optimal pharmacokinetic (PK) properties. To achieve an optimal balance between efficacy and toxicity due to cytokine release syndrome (CRS), the inventors fine-tuned the affinity of the anti-CD3 antibody and optimized tumor-associated antigen (TAA) binding. To increase the in vivo half-life (T1 / 2), a bifunctional linker was used to simultaneously conjugate both folate molecules and PEG molecules of various sizes. The optimized conjugate showed potent and selective in vitro activity, good serum half-life, and potent in vivo activity in a xenograft mouse model. This semi-synthetic approach is likely applicable to the generation of additional anti-CD3 bispecific agents using small molecule ligands selective for other TAAs.

[0012] The present invention provides an anti-CD3 antibody and a complex of the anti-CD3 antibody and folate. The present invention also provides an anti-CD3 antibody and a complex of the anti-CD3 antibody and PEG. The present invention also provides an anti-CD3 antibody and a complex of the anti-CD3 antibody, folate and PEG. In some embodiments, the novel anti-CD3 antibody of the present invention contains one or more non-naturally encoded amino acids. In some embodiments, the anti-CD3 antibody contains a complete antibody heavy chain. In some embodiments, the anti-CD3 antibody contains a complete antibody light chain. In some embodiments, the anti-CD3 antibody contains a complete antibody heavy chain and a complete antibody light chain. In some embodiments, the anti-CD3 antibody contains the variable region of the antibody light chain. In some embodiments, the anti-CD3 antibody contains the variable region of the antibody heavy chain. In some embodiments, the anti-CD3 antibody contains the variable region of the light chain and the variable region of the heavy chain. In some embodiments, the anti-CD3 antibody contains one or more CDRs of the antibody light chain. In some embodiments, the anti-CD3 antibody contains one or more CDRs of the anti-CD3 antibody heavy chain. In some embodiments, the anti-CD3 antibody contains one or more CDRs of the light chain and one or more CDRs of the heavy chain. In some embodiments, the anti-CD3 antibody contains three CDRs of the light chain. In some embodiments, the anti-CD3 antibody contains three CDRs of the heavy chain. In some embodiments, the anti-CD3 antibody contains three CDRs of the light chain and three CDRs of the heavy chain. In some embodiments, the anti-CD3 antibody contains Fab. In some embodiments, the anti-CD3 antibody contains two Fabs. In some embodiments, the anti-CD3 antibody contains two or more Fabs. In some embodiments, the anti-CD3 antibody contains scFv. In some embodiments, the anti-CD3 antibody contains two scFvs. In some embodiments, the anti-CD3 antibody contains two or more scFvs. In some embodiments, the anti-CD3 antibody contains a minibody. In some embodiments, the anti-CD3 antibody contains two minibodies. In some embodiments, the anti-CD3 antibody contains two or more minibodies. In some embodiments, the anti-CD3 antibody contains a diabody.In some embodiments, the anti-CD3 antibody comprises two diabodies. In some embodiments, the anti-CD3 antibody comprises two or more diabodies. In some embodiments, the anti-CD3 antibody comprises a BiTE. In some embodiments, the anti-CD3 antibody comprises two BiTEs. In some embodiments, the anti-CD3 antibody comprises two or more BiTEs. In some embodiments, the anti-CD3 antibody comprises a DART. In some embodiments, the anti-CD3 antibody comprises two DARTs. In some embodiments, the anti-CD3 antibody comprises two or more DARTs. In some embodiments, the anti-CD3 antibody comprises a TandAb. In some embodiments, the anti-CD3 antibody comprises two TandAbs. In some embodiments, the anti-CD3 antibody comprises two or more TandAbs. In some embodiments, the anti-CD3 antibody comprises a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the anti-CD3 antibody comprises a complete light chain and a complete heavy chain. In some embodiments, the anti-CD3 antibody comprises one or more Fc domains or portions thereof. In some embodiments, the anti-CD3 antibody comprises any combination of the above-described embodiments. In some embodiments, the anti-CD3 antibody comprises a homodimer, heterodimer, homotrimer, or heterotrimer of any of the above-described embodiments. In some embodiments, the anti-CD3 antibody comprises a polypeptide that binds to a binding target (wherein the binding target includes an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance). In some embodiments, the anti-CD3 antibody is bound to a scaffold molecule or substance other than an antibody.

[0013] In some embodiments, the anti-CD3 antibody has one or more post-translational modifications. In some embodiments, the anti-CD3 antibody is linked to a linker, a polymer, or a molecule having biological activity. In some embodiments, the anti-CD3 antibody is linked to a bifunctional polymer, a bifunctional linker, or at least one additional anti-CD3 antibody. In some embodiments, the anti-CD3 antibody is linked to a polypeptide that is not an anti-CD3 antibody. In some embodiments, an antigen-binding polypeptide having non-naturally encoded amino acids is linked to one or more additional antigen-binding polypeptides (the additional antigen-binding polypeptides may also have non-naturally encoded amino acids). In some embodiments, an antigen-binding polypeptide having non-naturally encoded amino acids is linked to one or more polypeptide-small molecule complexes (the polypeptide-small molecule complexes may also have non-naturally encoded amino acids). In some embodiments, an anti-CD3 antibody having non-naturally encoded amino acids is linked to one or more additional antigen-binding polypeptides (the additional antigen-binding polypeptides may also have non-naturally encoded amino acids).

[0014] In some embodiments, the non-naturally encoded amino acid is linked to a small molecule ligand. In some embodiments, the non-naturally encoded amino acid is linked to two small molecule ligands. In some embodiments, the non-naturally encoded amino acid is linked to two or more small molecule ligands. In some embodiments, the small molecule ligand has a folic acid molecule or a DUPA molecule. In some embodiments, the small molecule ligand has two folic acid molecules or two DUPA molecules. In some embodiments, the small molecule ligand has two or more folic acid molecules or two or more DUPA molecules. In some embodiments, the non-naturally encoded amino acid is linked to a water-soluble polymer. In some embodiments, the water-soluble polymer contains a poly(ethylene glycol) moiety. In some embodiments, the poly(ethylene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is an antigen-binding polypeptide. In some embodiments, the second polypeptide is an anti-CD3 antibody. In some embodiments, the small molecule ligand is linked to a water-soluble polymer. In some embodiments, two small molecule ligands are linked to two water-soluble polymers. In some embodiments, two or more small molecule ligands are linked to two or more water-soluble polymers. In some embodiments, the folate is linked to a PEG molecule. In some embodiments, two folic acid molecules are linked to two water-soluble polymers. In some embodiments, two or more folic acid molecules are linked to two or more PEG molecules.

[0015] In some embodiments, the amino acid substitution in the anti-CD3 antibody may be by an amino acid that occurs naturally or by an amino acid that does not occur naturally. However, at least one substitution is by a non-naturally encoded amino acid.

[0016] In some embodiments, the non-naturally encoded amino acids have a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group.

[0017] In some embodiments, the average molecular weight of the poly(ethylene glycol) molecule is from about 0.1 kDa to about 100 kDa. In some embodiments, the average molecular weight of the poly(ethylene glycol) molecule is from 0.1 kDa to 50 kDa. In some embodiments, the average molecular weight of the poly(ethylene glycol) is from 1 kDa to 25 kDa, from 2 kDa to 22 kDa, or from 5 kDa to 20 kDa. For example, the average molecular weight can be about 5 kDa, or about 10 kDa, or about 20 kDa. For example, the average molecular weight of the poly(ethylene glycol) polymer can be 5 kDa, or 10 kDa, or 20 kDa. In certain embodiments, the molecular weight is measured by a suitable method such as SDS / PAGE analysis, RP-HPLC, SEC, mass spectrometry, and capillary electrophoresis.

[0018] In some embodiments, the poly(ethylene glycol) molecule is a branched polymer. In some embodiments, the molecular weight of each branch of the poly(ethylene glycol) branched polymer is from 1 kDa to 100 kDa, or from 1 kDa to 50 kDa. In some embodiments, the molecular weight of each branch of the poly(ethylene glycol) branched polymer is from 1 kDa to 25 kDa, from 2 kDa to 22 kDa, or from 5 kDa to 20 kDa. For example, the molecular weight of each branch of the poly(ethylene glycol) branched polymer can be about 5 kDa, or about 10 kDa, or about 20 kDa. For example, the molecular weight of each branch of the poly(ethylene glycol) branched polymer can be 5 kDa, or 10 kDa, or 20 kDa.

[0019] The present invention also provides an anti-CD3 antibody polypeptide comprising a linker, polymer or molecule having biological activity, which is bound to one or more non-naturally encoded amino acids (the non-naturally encoded amino acids are incorporated by ribosomes at a preselected position of the polypeptide).

[0020] Embodiments of the present invention provide anti-CD3 antibodies having one or more of SEQ ID NOs: 1 to 62. An anti-CD3 antibody having two of SEQ ID NOs: 1 to 62. Embodiments of the present invention provide an anti-CD3 antibody having two of SEQ ID NOs: 1 to 62. An anti-CD3 antibody having any one of SEQ ID NOs: 1 to 6 and any one of SEQ ID NOs: 7 to 9. Embodiments of the present invention provide an anti-CD3 Fab antibody having two of SEQ ID NOs: 1 to 5. Another embodiment of the present invention provides a bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain is selected from the group consisting of SEQ ID NOs: 1 to 62. Another embodiment of the present invention provides a bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain has any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57, and any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62, and comprises an anti-CD3. Another embodiment of the present invention provides a bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain has any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57, and comprises an anti-CD3 Fab.Other embodiments of the present invention are bispecific binding molecules having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain is any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57; and any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62, and providing a bispecific binding molecule comprising an anti-CD3 Fab. Other embodiments of the present invention provide a cytotoxicity-active CD3-specific binding construct having an amino acid sequence set forth in one or more of SEQ ID NOs: 1-62. Other embodiments of the present invention provide a cytotoxicity-active CD3-specific binding construct having an amino acid sequence set forth in two of SEQ ID NOs: 1-62. Other embodiments of the present invention provide a cytotoxicity-active CD3-specific binding construct comprising an anti-CD3 having any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57. Other embodiments of the present invention provide a cytotoxicity-active CD3Fab-specific binding construct comprising an anti-CD3 Fab having any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62.Other embodiments of the present invention provide a cytotoxic, CD3-specific binding construct comprising an anti-CD3 having any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57; and any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. Other embodiments of the present invention provide an anti-CD3 Fab antibody, wherein the anti-CD3 antibody has a binding domain comprising (a) a VH domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and (b) a VL domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9.

[0021] Other embodiments of the present invention provide an anti-CD3 antibody, wherein the antibody comprises one or more post-translational modifications. Other embodiments of the present invention provide an anti-CD3 Fab antibody, wherein the antibody is linked to a linker, a polymer, or a molecule having biological activity. Other embodiments of the present invention provide an anti-CD3 antibody, wherein the molecule having biological activity is folate. Embodiments of the present invention provide an anti-CD3 antibody comprising one or more folates. Embodiments of the present invention provide an anti-CD3 antibody having two folates. Other embodiments of the present invention provide an anti-CD3 antibody, wherein the molecule having biological activity is DUPA. Embodiments of the present invention provide an anti-CD3 antibody comprising one or more DUPAs. Embodiments of the present invention provide an anti-CD3 antibody having two DUPAs.

[0022] In certain embodiments, the anti-CD3 antibody has any one of the heavy chain amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57; and any one of the light chain amino acid sequences of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62.

[0023] In some embodiments, the anti-CD3 antibody comprises an anti-CD3 having any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 having any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62.

[0024] In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57; and any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. Other embodiments of the present invention provide an anti-CD3 variant having a heavy chain of SEQ ID NOs: 1-5 and a light chain of SEQ ID NOs: 7-9. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 7 and 10, or 7 and 14, or 7 and 11, or 7 and 15, or 7 and 12, or 7 and 16, or 7 and 13, or 7 and 17, or 7 and 1, or 7 and 18, or 7 and 19, or 12 and 18, or 12 and 19 or 12 and 16. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 7 and 10, or 7 and 14, or 7 and 11, or 7 and 15, or 7 and 12, or 7 and 16, or 7 and 13, or 7 and 17, or 7 and 1, or 7 and 18, or 7 and 19, or 12 and 18, or 12 and 19 or 12 and 16, each of which sequences contains one or more non-natural amino acids. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 7 and 10, or 7 and 14, or 7 and 11, or 7 and 15, or 7 and 12, or 7 and 16, or 7 and 13, or 7 and 17, or 7 and 1, or 7 and 18, or 7 and 19, or 12 and 18, or 12 and 19 or 12 and 16, each of which sequences contains two non-natural amino acids.In some embodiments, the anti-CD3 antibody comprises SEQ ID NO: 7 containing one or more non-natural amino acids; and an anti-CD3 Fab having one of SEQ ID NO: 10 or 14 or 11 or 15 or 12 or 16 or 13 or 17 or 1 or 18 or 19 containing one or more non-natural amino acids. In some embodiments, the anti-CD3 antibody comprises SEQ ID NO: 12 containing one or more non-natural amino acids; and an anti-CD3 Fab having one of SEQ ID NO: 18 or 19 or 16 containing one or more non-natural amino acids. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 58 and 49, or 58 and 40, or 59 and 50, or 59 and 41, or 59 and 42, or 59 and 52, or 59 and 43, or 59 and 44, or 59 and 45 or 59 and 54 or 59 and 55, or 59 and 46, or 9 and 44, or 9 and 53, or 60 and 44, or 60 and 53, or 60 and 42, or 60 and 51, 60 and 50, or 60 and 41, or 61 and 45, or 61 and 54, or 62 and 56, or 62 and 47, or 62 and 48, or 62 and 57, or 7 and 47, or 7 and 56. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having two non-natural amino acids, and the antibody has SEQ ID NO: 18 and 16, or 18 and 59, or 18 and 43.

[0025] In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 1 and 7, or 1 and 8, or 1 and 9. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 2 and 7, or 2 and 8, or 2 and 9. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 3 and 7, or 3 and 8, or 3 and 9. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 4 and 7, or 4 and 8, or 4 and 9. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 5 and 7, or 5 and 8, or 5 and 9. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 6 and 7, or 6 and 8, or 6 and 9. The anti-CD3 antibody is a bispecific antibody having (i) a first binding domain and (ii) a second binding domain, and the second binding domain may comprise the anti-CD3 Fab. Other embodiments of the present invention provide anti-CD3 Fab antibodies selected from SEQ ID NOs: 1-62 into which non-naturally encoded amino acids are incorporated. In some embodiments, the non-naturally encoded amino acids are site-specifically incorporated into the antibody.

[0026] Other embodiments provide anti-CD3 Fab antibodies in which the non-naturally encoded amino acid is selected from the group consisting of O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, p-propargyloxy-L-phenylalanine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

[0027] Other embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at any one of the positions of H114, H115, H129, L157, H160, L172, and L205 (according to the Kabat numbering well-known to those skilled in the art). Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at the positions of H114, H115, H129, L157, H160, L172, and L205 according to Kabat numbering. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 114. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 115. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 129. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 157. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 160. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 172. Embodiments of the present invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are site-specifically incorporated at position 205. Other embodiments of the present invention provide anti-CD3 Fab antibodies in which one or more non-naturally encoded amino acids are incorporated. Other embodiments of the present invention provide anti-CD3 Fab antibodies in which two non-naturally encoded amino acids are incorporated. Other embodiments of the present invention provide anti-CD3 Fab variants comprising one or more non-naturally encoded amino acids in the heavy or light chain.Other embodiments of the invention provide anti-CD3 Fab antibodies that include one non-naturally encoded amino acid in the light chain. Other embodiments of the invention provide anti-CD3 Fab antibodies that include one non-naturally encoded amino acid in the heavy chain. Other embodiments of the invention provide anti-CD3 Fab variants that include two non-naturally encoded amino acids.

[0028] Other embodiments of the invention provide anti-CD3 Fab variants in which the heavy chain further includes an amino acid extension at the C-terminus. Embodiments of the invention provide anti-CD3 Fab variants in which the amino acid extension includes the amino acids DKTHT. Other embodiments of the invention provide anti-CD3 Fab variants that further include an amino acid extension in the C-terminal heavy chain. Other embodiments of the invention provide anti-CD3 Fab variants in which the amino acid extension includes the amino acids DKTHT.

[0029] Other embodiments of the invention provide anti-CD3 Fab antibodies in which the heavy chain sequence has mouse framework residues at one or more positions for antigen binding. Other embodiments of the invention provide anti-CD3 Fab antibodies in which the heavy chain sequence has mouse framework residues at position 30, 49, 77, or 93 (by Kabat numbering). Other embodiments of the invention provide anti-CD3 Fab antibodies in which the light chain sequence has mouse framework residues at one or more positions. Other embodiments of the invention provide anti-CD3 Fab antibodies in which the light chain sequence has framework residues at position 36, 46, 49, 57, or 58 (by Kabat numbering).

[0030] In some embodiments of the present invention, the anti-CD3 Fab antibody comprises a linker. Other embodiments of the present invention provide an anti-CD3 Fab antibody wherein the linker is a water-soluble polymer. Other embodiments of the present invention provide an anti-CD3 Fab antibody wherein the water-soluble polymer comprises poly(ethylene glycol). Other embodiments of the present invention provide an anti-CD3 Fab antibody wherein the water-soluble polymer is a linear or branched water-soluble polymer. Other embodiments of the present invention provide an anti-CD3 Fab antibody wherein the water-soluble polymer is linked to an amino acid not naturally encoded in the antibody. In some embodiments, the linker is bound to the anti-CD3 Fab antibody via the side chain of a non-natural amino acid. Embodiments of the present invention provide an anti-CD3 Fab antibody comprising two or more amino acids linked to a water-soluble polymer comprising poly(ethylene glycol). Other embodiments of the present invention provide an anti-CD3 Fab antibody wherein one or more of the amino acids linked to the water-soluble polymer are amino acids not naturally encoded. Other embodiments of the present invention provide an anti-CD3 Fab antibody comprising one or more poly(ethylene glycols). Other embodiments of the present invention provide an anti-CD3 Fab antibody wherein the poly(ethylene glycol) is from 1 kDa to 100 kDa. Other embodiments of the present invention provide an anti-CD3 Fab antibody comprising one or more folates and one or more poly(ethylene glycols). Other embodiments of the present invention provide an anti-CD3 Fab antibody comprising two folates and two poly(ethylene glycols).

[0031] Other embodiments of the invention are methods of optimizing cell killing in cells that highly express folate receptor, comprising an anti-CD3 Fab antibody, said antibody comprising one or more folates, and one or more non-naturally encoded amino acids incorporated into said antibody. Other embodiments of the invention are methods of optimizing the killing of tumor cells that highly express folate receptor by redirected T cells, comprising an anti-CD3 Fab antibody, said antibody comprising one or more folates, and one or more non-naturally encoded amino acids incorporated into said antibody. In other embodiments, the method further comprises one or more water-soluble polymers. Other embodiments of the invention provide a method wherein the water-soluble polymer comprises poly(ethylene glycol). Additionally, the water-soluble polymer is a linear or branched water-soluble polymer. The poly(ethylene glycol) is from 1 kDa to 100 kDa. The poly(ethylene glycol) is 5, 10, 20, 30, 40, 50, or 60 kDa. In other embodiments, the number of folate receptors is 10,000 or more.

[0032] Other embodiments of the invention provide methods for reducing cytotoxicity in cells. Other embodiments of the invention provide methods for reducing cytotoxicity in cells by optimizing or reducing the affinity of an anti-CD3 antibody for a target for T cell accumulation. Other embodiments of the invention provide methods for enhancing the cytotoxicity of tumor cells by complexing two or more folates to preferentially bind to tumor cells prior to T cell binding. Other embodiments of the invention provide methods for improving the serum half-life of an anti-CD3 Fab antibody. Other embodiments of the invention provide methods for improving the serum half-life of an anti-CD3 Fab antibody by conjugating the Fab to a pharmacokinetic (PK) extender molecule (including but not limited to HSA, C12-C16 acyl chains, XTEN, or a water-soluble polymer such as PEG). Other embodiments provide methods for accumulating cytotoxic T cells in FR+ tumor cells. Other embodiments provide methods for improving efficacy, reducing toxicity, improving PK properties, improving affinity, improving TAA binding, improving in vivo T1 / 2, improving in vitro activity, improving serum half-life, and / or improving in vivo activity. Other embodiments provide methods for improving efficacy. Other embodiments provide methods for reducing toxicity. Other embodiments provide methods for improving PK properties. Other embodiments provide methods for improving affinity. Other embodiments provide methods for improving TAA binding. Other embodiments provide methods for improving in vivo T1 / 2. Other embodiments provide methods for improving in vitro activity. Other embodiments provide methods for improving in vivo activity.

[0033] Other embodiments of the invention provide anti-CD3 Fab antibodies in which the heavy chain sequence or the light chain sequence has a human germline mutation at one or more positions. Embodiments of the invention provide anti-CD3 Fab antibodies in which the human germline mutation in the heavy chain sequence is located at position 35 or 52 (by Kabat numbering). Other embodiments of the invention provide anti-CD3 Fab antibodies in which the human germline mutation in the light chain sequence is located at position 53 (by Kabat numbering).

[0034] Other embodiments of the invention provide an anti-CD3 Fab antibody further comprising a PEG-folate linker having the structures of compounds 29A, 29B, 29C, 29D, 29E, 30A, 30B, 30C, 30D and 30E.

[0035] Other embodiments provide a method of treating a patient having a disease or condition in cells that highly express the folate receptor, the method comprising administering to the patient a therapeutically effective amount of the anti-CD3 Fab antibody described herein. Other embodiments of the invention provide a bispecific anti-CD3 Fab comprising two folate molecules and two PEGylated molecules. In other embodiments, the bispecific anti-CD3 Fab antibody further comprises non-naturally encoded amino acids site-specifically incorporated therein. The present invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is an epithelial tumor, a stromal tumor, and a germ cell tumor. In some embodiments, the ovarian cancer includes fallopian tube cancer and primary peritoneal carcinoma. In some embodiments, the cancer is characterized by high expression of folate receptor α (FOLR1) and is ovarian cancer or the like. In some embodiments, the cancer is treated by accumulating cytotoxic T cells in folate receptor positive (FR+) tumor cells. The present invention provides a method of treating a genetic disease by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. The present invention provides a method of treating AIDS by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. The present invention provides a method of treating diabetes by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. The anti-CD3 antibody of the invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, which optionally is complexed with two folate molecules and two PEGylated molecules. In further embodiments, non-naturally encoded amino acids are site-specifically incorporated into the anti-CD3 Fab antibody and are complexed with two folate molecules and two PEGylated molecules via the side chains of the non-natural amino acids.

[0036] The anti-CD3 antibody of the present invention is used for the treatment of diseases or medical conditions in cells that highly express the folate receptor. The anti-CD3 antibody of the present invention is used for the treatment of cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is an epithelial tumor, a stromal tumor, and a germ cell tumor. In some embodiments, the ovarian cancer includes fallopian tube cancer and primary peritoneal carcinoma. In some embodiments, the cancer is characterized by high expression of folate receptor α (FOLR1), such as ovarian cancer. In some embodiments, the cancer is treated by accumulating cytotoxic T cells in folate receptor-positive (FR+) tumor cells. The anti-CD3 antibody of the present invention is used for the manufacture of a medicament for the treatment of genetic diseases. The anti-CD3 antibody of the present invention is used for the treatment of AIDS. The anti-CD3 antibody of the present invention is used for the treatment of diabetes. The above anti-CD3 antibody of the invention is a bispecific antibody comprising an anti-CD3 Fab antibody, and the anti-CD3 Fab antibody can optionally be a bispecific antibody complexed with two folic acid molecules and two PEGylated molecules. In a further embodiment, non-naturally encoded amino acids are site-specifically incorporated into the anti-CD3 Fab antibody and are complexed with two folic acid molecules and two PEGylated molecules via the side chains of the non-natural amino acids. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating diseases or medical conditions in cells that highly express the folate receptor. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is an epithelial tumor, a stromal tumor, and a germ cell tumor. In some embodiments, the ovarian cancer includes fallopian tube cancer and primary peritoneal carcinoma. In some embodiments, the cancer is characterized by high expression of folate receptor α (FOLR1), such as ovarian cancer. In some embodiments, the cancer is treated by accumulating cytotoxic T cells in folate receptor-positive (FR+) tumor cells. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating genetic diseases. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating AIDS. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating diabetes.The above anti-CD3 antibody of the invention is a bispecific antibody comprising an anti-CD3 Fab antibody, and the anti-CD3 Fab antibody can optionally be a bispecific antibody complexed with two folic acid molecules and two PEGylated molecules. In a further embodiment, non-naturally encoded amino acids are site-specifically incorporated into the anti-CD3 Fab antibody and are complexed with two folic acid molecules and two PEGylated molecules via the side chains of the non-natural amino acids.

[0037] The disclosure of the present invention provides an anti-CD3 antibody or antibody fragment or variant having one or more non-naturally encoded amino acids incorporated therein. Anti-CD3 antibodies, antibody fragments, or variants include, but are not limited to, Fv, Fc, Fab and (Fab’)2, single-chain Fv (scFv), diabody, triabody, tetrabody, bifunctional hybrid antibody, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules having other backbones, bispecific antibodies, and the like. In some embodiments, the anti-CD3 antibody or antibody fragment or variant is an anti-CD3 Fab antibody, fragment, or variant.

[0038] The disclosure of the invention provides a bispecific antibody complex comprising an anti-CD3 antibody or antibody fragment. The invention discloses a bispecific antibody complex comprising an anti-CD3 Fab antibody or antibody fragment into which one or more non-naturally encoded amino acids are incorporated. Further described is a bispecific antibody complex comprising an anti-CD3 Fab antibody or antibody fragment and one or more folic acid molecules, wherein one or more non-naturally encoded amino acids are site-specifically incorporated into the anti-CD3 Fab antibody. Described is a bispecific antibody complex comprising an anti-CD3 Fab antibody or antibody fragment, one or more folic acid molecules, and one or more PEG molecules, wherein one or more non-naturally encoded amino acids are site-specifically incorporated into the anti-CD3 Fab antibody. Described is an anti-CD3 Fab bispecific antibody comprising an antibody or antibody fragment, one or more small molecules, and one or more linkers, wherein the antibody or antibody fragment is linked to the one or more small molecules by the one or more linkers, and the small molecules are one or more folic acid molecules or one or more DUPA molecules or analogs or derivatives. The antibody or antibody fragment can be site-specifically linked to one or more folic acid molecules or one or more DUPA molecules by one or more linkers. The antibody or antibody fragment can contain one or more non-natural amino acids. The antibody or antibody fragment can be linked to one or more folic acid molecules or one or more DUPA molecules by one or more linkers for one or more non-natural amino acids. The non-natural amino acids may be site-specifically incorporated into the antibody. The antibody or antibody fragment can be linked to one or more folic acid molecules or DUPA molecules by one or more PEG molecules for one or more non-natural amino acids. Alternatively, the antibody or antibody fragment can be linked to one or more folic acid molecules or one or more DUPA molecules by one or more linkers for natural amino acids. The antibody or antibody fragment can be an anti-CD3 Fab.

[0039] A bispecific antibody conjugate comprising an anti-CD3 Fab, one or more folic acid molecules, and one or more linkers, wherein the anti-CD3 Fab is linked to the one or more folic acid molecules by the one or more linkers, is described. The anti-CD3 Fab may comprise one or more non-natural amino acids. The one or more non-natural amino acids may replace the natural amino acids of the anti-CD3 Fab. A bispecific antibody conjugate comprising an anti-CD3 Fab, one or more DUPA molecules, and one or more linkers, wherein the anti-CD3 Fab is linked to the one or more DUPA molecules by the one or more linkers, is described. The anti-CD3 Fab may comprise one or more non-natural amino acids. The one or more non-natural amino acids may replace the natural amino acids of the anti-CD3 Fab.

[0040] [Brief Description of the Drawings] The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the disclosure of the invention will be obtained by reference to the following detailed description, which describes exemplary embodiments in which the principles of the invention are utilized, and to the appended drawings.

[0041] Figure 1 shows a humanized anti-CD3 Fab bound to human PBMC. Binding of humanized anti-CD3 Fab expressed in HEK293 cells to human PBMC (two experiments) is shown. A total of 9 Fabs were tested by combining 3vH chain sequences and 3vL chain sequences. Three Fabs with the vL1.0 chain lost binding to human CD3 in combination with all vH chains.

[0042] Figures 2A-2F show titrations of humanized anti-CD3 Fab bound to human PBMC and canine PBMC. (Figure 2A) Titration of Fab1; (Figure 2B) Titration of Fab2; (Figure 2C) Titration of Fab3; (Figure 2D) Titration of Fab4; (Figure 2E) Titration of Fab5; and (Figure 2F) Titration of Fab6.

[0043] Figure 3 shows the pharmacokinetic (PK) analysis of the anti-CD3 Fab1 molecule in rats. PEGylation of the humanized anti-CD3 Fab1 molecule extends the plasma half-life (T1 / 2) in rats.

[0044] Figures 4A - 4B show the binding of the first-round low-affinity Fabs derived from HEK293 cells. The first-round anti-CD3 Fab mutants generated from HEK293 cells with low binding affinities for human CD3 (Figure 4A) and canine CD3 (Figure 4B) are shown. Titration curves for four selected low-affinity Fabs (Fab7 - 10) and control Fab1 among 35 new Fab mutants tested in the first-round screening are shown.

[0045] Figures 5A - 5B show the binding of the second-round low-affinity Fabs derived from HEK293 cells to human CD3. The second-round anti-CD3 Fab mutants generated from HEK293 cells with low binding affinities for human CD3 (Figure 5A) and canine CD3 (Figure 5B) are shown. Titration curves for four selected low-affinity Fabs and parental control Fab1 among 43 new Fab mutants tested in the second-round screening are shown.

[0046] Figures 6A - 6B show the binding of the low-affinity Fab-folate mutants derived from E. coli cells to human CD3. Figure 6A shows the first-round binding of the low-affinity mutants of the anti-CD3 Fab-HK129-folate molecule purified from E. coli cells to human CD3, and Figure 6B shows the second-round binding. The parental control Fab1-HK129-folate is included as a positive control.

[0047] Figures 7A - 7B show the low-affinity mutants of anti-CD3 Fab-folate binding to canine CD3. Figure 7A shows the first-round binding of the low-affinity mutants of the anti-CD3 Fab-HK129-folate molecule purified from E. coli cells to canine CD3, and Figure 7B shows the second-round binding. The parental control Fab1-HK129-folate is included as a positive control.

[0048] Figure 8 shows the cytotoxicity to SKOV-3 cells with human PBMC. In vitro cytotoxicity of four low-affinity variants of the humanized anti-CD3 Fab-HK129-folic acid molecule generated from E. coli cells with human PBMC. The parental control Fab1-HK129-folate is included as a positive control.

[0049] Figure 9 shows the cytotoxicity to SKOV-3 cells with canine PBMC. In vitro cytotoxicity of four low-affinity variants of the humanized anti-CD3 Fab-HK129-folic acid molecule generated from E. coli cells with canine PBMC. The parental control Fab1-HK129-folate is included as a positive control.

[0050] Figures 10A - 10B show the activation of T cells. Figure 10A shows the activation of T cell markers CD25 and CD69 without SKOV-3 cells by various low-affinity anti-CD3 Fab-HK129-folic acid molecules. Figure 10B shows the activation of T cell markers CD25 and CD69 with SKOV-3 cells by various low-affinity anti-CD3 Fab-HK129-folic acid molecules.

[0051] Figures 11A - 11D show the in vitro cytokine release by IFNγ (Figures 11A and 11B), and the in vitro cytokine release by TNFα (Figures 11C and 11D) of various low-affinity anti-CD3 Fab-HK129-folic acid variants without SKOV-3 tumor cells (Figures 11A and 11C) or with SKOV-3 tumor cells (Figures 11B and 11D).

[0052] Figures 12A - 12F show the anti - CD3Fab - folic acid bispecific conjugate and PEG linker of the present invention. Figure 12A shows a representative example of a folic acid - PEG ligand; Figure 12B shows a representative example of a folic acid - branched PEG ligand; Figures 12C - 12D show representative examples of CD3 - folic acid bispecifics containing a PEG conjugate; Figure 12E shows a representative example of a CD3 - folic acid bispecific containing PEGylation at the C - terminus; Figure 12F shows a representative example of a CD3 - folic acid bispecific containing PEGylation by CD3 - Fab cross - linking at the C - terminus.

[0053] Figures 13A - 13D show CD3Fab - folic acid conjugates with PEGylation and CD3Fab - folic acid conjugates without PEGylation. Figures 13A and 13B show SDS - PAGE gel electrophoresis analysis of anti - CD3Fab monomer composition and anti - CD3Fab double - complex composition; Figure 13C shows SDS - PAGE gel electrophoresis analysis of anti - CD3Fab - folic acid C - terminal PEG conjugate; Figure 13D shows the in vitro cytotoxicity of anti - CD3Fab - folic acid C - terminal PEG conjugate in KB cells, OV - 90 cells, and SKOV - 3 cells.

[0054] Figure 14 shows an anti - CD3Fab - folic acid bispecific antibody that shows in vitro cytotoxicity and selectively kills FOLRα - expressing KB cells.

[0055] Figures 15A - 15B show in vitro cytotoxicity. Figure 15A shows an anti - CD3Fab - folic acid bispecific antibody that selectively kills FOLRα - expressing SKOV3 cells in the presence of 20 nM folic acid, and Figure 15B shows selective killing in the presence of 50 nM folic acid.

[0056] Figures 16A - 16B show in vitro cytotoxicity data and show an anti - CD3Fab - folic acid bispecific antibody that selectively kills FOLRα - expressing SKOV3 cells in the presence of 20 nM 5 - mTHF (Figure 16A) and 50 nM 5 - mTHF (Figure 16B).

[0057] Figure 17 shows a mouse pharmacokinetic study in CD1 mice.

[0058] Figures 18A-18B show an anti-CD3Fab-folate bispecific antibody that selectively kills human M2 macrophages. Figure 18A shows in vitro macrophage cytotoxicity by a single folate salt containing the anti-CD3Fab-folate bispecific antibody. Arrows and numbers represent the difference (magnification) between M1 macrophages and M2 macrophages at IC 50 . The dotted line indicates Fab1-HK129-folate in M1 and M2, and the solid line indicates Fab1-HK129-5KPEG-folate in M1 and M2. Figure 18B shows in vitro macrophage cytotoxicity by a dual folate salt containing the anti-CD3Fab-folate bispecific antibody. Arrows and numbers represent the difference (magnification) between M1 macrophages and M2 macrophages at IC 50 . The dotted line indicates Fab1-HK129-LL157-BiFolate in M1 and M2, and the solid line indicates Fab1-HK129-LL157-BiFolate-Bi5KPEG in M1 and M2.

[0059] Figures 19A-19B show the antitumor efficacy of an anti-CD3-folate bispecific antibody. Figure 19A shows tumor volume, and Figure 19B shows mass / body weight. Figures 19C and 19D show the expression of human CD45 and the induction of TIL, respectively.

[0060] Figures 20A-20B show the antitumor efficacy of an anti-CD3-folate bispecific antibody. Figure 20A shows tumor volume, and Figure 20B shows mass / body weight. Figures 20C and 20D show the expression of human CD45 and the induction of TIL, respectively.

[0061] Figures 21A-21F show the antitumor efficacy by multiple administrations of a CD3-folate bispecific antibody in a human cervical tumor derived from a KB cell line (Figure 21A shows tumor growth, and Figure 21B shows body weight), and the expression of T cell activation markers CD25 (Figure 21C), CD69 (Figure 21D), CD45 (Figure 21E), and CD3 (Figure 21F).

[0062] Figures 22A-22B show the antitumor efficacy of the CD3-folate bispecific antibody in human cervical tumors derived from the KB cell line; Figure 22A shows tumor growth and Figure 22B shows body weight.

[0063] Figures 23A-23B show the antitumor efficacy of the CD3-folate bispecific antibody compared to carboplatin in human ovarian tumors derived from the OV-90 cell line; Figure 23A shows tumor growth and Figure 23B shows body weight.

[0064] 〔Definitions〕 It should be understood that the present invention is not limited to the specific methods, protocols, cell lines, compositions, and agents described herein, but can be modified. It should also be understood that the terms used herein are only used to describe specific embodiments and are not intended to limit the scope of the present invention. The scope of the present invention is limited only by the appended claims.

[0065] As used in this specification and the appended claims, the singular forms ( "a", "an", "the") include plural referents unless the context clearly dictates otherwise. Thus, for example, references to "anti-CD3 Fab" or "anti-CD3 Fab-folate antibody" are references to one or more of such proteins, including equivalents known to those skilled in the art. The terms "PEG-folate" or "folate-PEG" and the terms "BiPEG-BiFolate" or "BiFolate-BiPEG" are used interchangeably herein.

[0066] 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 this invention belongs. Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. However, the preferred methods, devices, and materials are those described herein.

[0067] Publications and patents mentioned in this specification are hereby incorporated by reference herein. This is for the purpose of, for example, explaining and disclosing the configurations and methods described in the publications that can be used in the context of the present invention. The publications discussed herein are merely for providing the disclosure prior to the filing date of the present application. Any statement in this specification should not be construed as an admission that the inventors are not entitled to antedate the above disclosure on the basis of prior invention or other reasons.

[0068] The term "substantially purified" means that the anti-CD3 Fab antibody is substantially or essentially free of components that are normally associated with or interact with proteins as found in the natural production environment (i.e., natural cells) or host cells (in the case of recombinantly produced anti-CD3 antibodies). Examples of anti-CD3 antibodies that are substantially free of cellular material include preparations containing less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of contaminating protein. When the anti-CD3 antibody or its variant is produced recombinantly from a host cell, such proteins may be present at about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the dry weight of the cell. When the anti-CD3 antibody or its variant is produced recombinantly from a host cell, such proteins may be present in the periplasm and / or medium at about 5 g / L or less, about 4 g / L or less, about 3 g / L or less, about 2 g / L or less, about 1 g / L or less, about 750 mg / L or less, about 500 mg / L or less, about 250 mg / L or less, about 100 mg / L or less, about 50 mg / L or less, about 10 mg / L or less, or about 1 mg / L or less of the dry weight of the cell. Thus, when provided by the methods of the present invention, the purity of the "substantially purified" anti-CD3 antibody may be about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more. Specifically, the purity may be about 75% or more, about 80% or more, about 85% or more. More specifically, the purity may be about 90% or more, about 95% or more, about 99% or more, or greater. Here, the purity is determined by an appropriate method (such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis).

[0069] "Recombinant host cell" or "host cell" refers to a cell containing an exogenous polynucleotide. The method of inserting the exogenous polynucleotide is not limited (for example, direct uptake, transformation, transduction, f mating, or other methods known in the technical field related to the production of recombinant host cells). The exogenous polynucleotide may be maintained as a non-integrated vector (such as a plasmid), or alternatively, it may be integrated into the host genome.

[0070] An antibody is a protein that exhibits binding specificity to a specific antigen. A natural antibody is usually a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains (L chains) and two identical heavy chains (H chains). Each light chain is linked to the heavy chain by a single covalent disulfide bond. The number of disulfide bonds between the heavy chains varies depending on the immunoglobulin isotype. In addition, each heavy chain and each light chain have disulfide cross-links between the chains at regular intervals. At one end of each heavy chain, there is a variable domain (V H ) followed by several constant domains. At one end of each light chain, there is a variable domain (V L ) and at the other end, there is a constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain. Also, the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain.

[0071] The term "variable" indicates that the sequences of specific portions of the variable domains vary significantly among antibodies. These sequence differences are responsible for the binding specificities of each particular antibody to a specific antigen. However, this variability is not evenly distributed throughout the variable domains of the antibody. The variability is concentrated in three portions called complementarity-determining regions (CDRs), which are present in both the light-chain variable domain and the heavy-chain variable domain. The more conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions. Many of them adopt a β-sheet structure and are linked to the three CDRs. The CDRs form loop structures and in some cases form part of the β-sheet structure. The CDRs of each chain are grouped together in close proximity by the FR regions. And together with the CDRs of the other chain, they contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0072] Constant domains do not directly participate in the binding of an antibody to its antigen but exhibit various effector functions. Antibodies or immunoglobulins can be classified into different classes according to the amino acid sequence of the heavy-chain constant region. There are five major classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM), some of which can be further classified into subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2). The heavy-chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light-chain constant regions corresponding to different classes of immunoglobulins are called κ and λ, respectively. Among the various classes of human immunoglobulins, only human IgG1, human IgG2, human IgG3, and human IgM are known to activate complement. An immunoglobulin can be selected from IgG, IgA, IgD, IgE, IgM, or fragments or modifications thereof.

[0073] Affinity maturation of antibodies in vivo proceeds mainly by antigen selection of high-affinity antibody variants generated mainly by somatic hypermutation. Also, usually, a "repertoire shift" occurs. In a repertoire shift, it has been found that the major germline genes of the secondary or tertiary responses are different from those of the primary and secondary responses.

[0074] The process of affinity maturation of the immune system can be reproduced by introducing mutations into antibody genes in vitro and isolating mutants with improved affinity by affinity selection. Such mutant antibodies can be displayed on the surface of filamentous bacteriophages or microorganisms (such as Escherichia coli, yeast). Antibodies can be selected based on their affinity for antigens or their dissociation rate (off-rate) from antigens (Hawkins et al. J. Mol. Biol. 226:889-896 (1992)). There are examples of affinity maturation of human antibodies that bind to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) by CDR walking mutagenesis (Barbas III et al. PNAS (USA) 91: 3809-3813 (1994); Yang et al. J. Mol. Biol. 254:392-403 (1995)). There are also examples of affinity maturation of anti-c-erbB-2 single-chain Fv fragments by a similar method (Schier et al. J. Mol. Biol. 263:551567 (1996)). There are examples of affinity maturation of human antibodies with high affinity for the third hypervariable loop of HIV by antibody chain shuffling and CDR mutagenesis (Thompson et al. J. Mol. Biol. 256:77-88 (1996)). [Balint and Larrick Gene 137:109-118 (1993)] discloses computer-assisted oligodeoxyribonucleotide-specific scanning mutagenesis. In this method, the entire CDR of the variable region gene is searched simultaneously to obtain improved mutants. There are also examples of affinity maturation of humanized antibodies specific for αvβ3 by the initial limited mutagenesis strategy. In this method, mutations are introduced at all positions of the six CDRs and expressed to obtain a combinatorial library containing mutants with the maximum affinity by screening (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)).Reviews of antibodies presented on phage are available in [Chiswell and McCafferty TIBTECH 10:80-84 (1992); Rader and Barbas III Current Opinion in Biotech. 8:503-508 (1997)]. In any of the examples reported in the above-mentioned literature where mutant antibodies with improved affinity compared to the parental antibody were reported, the mutant antibodies had amino acid substitutions in the CDRs.

[0075] As used herein, "affinity maturation" means a process of improving the affinity of an antibody for an antigen. Methods of affinity maturation include, but are not limited to, computer screening methods and experimental methods.

[0076] As used herein, "antibody" means a protein comprising one or more polypeptides substantially encoded by all or part of an antibody gene. Immunoglobulin genes include, but are not limited to, the constant region genes kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3, and IgG4), delta, epsilon, and mu, as well as the myriad immunoglobulin variable region genes. Antibodies herein include full-length antibodies and antibody fragments. Also included are antibodies that occur naturally in any organism, as well as artificially produced antibodies (e.g., variants). The antibodies described herein can be human antibodies, humanized antibodies, engineered antibodies, non-human antibodies, and / or chimeric antibodies. Humanized antibodies and methods for producing such are well known in the art. (See, e.g., U.S. Patent Nos. 5,821,337; 7,527,791; 6,982,321; 7,087,409; 5,766,886). Generally, a humanized antibody contains one or more variable domains in which the CDR or a portion thereof is derived from a non-human antibody and the framework region or a portion thereof is derived from a human antibody sequence. A humanized antibody optionally can contain at least a portion of a human constant region. In some embodiments, for example, framework residues in a humanized antibody can be substituted with the corresponding residues from a non-human antibody (the antibody from which the CDR residues are derived) to restore or improve antibody specificity or antibody affinity. A chimeric antibody can refer to an antibody produced by combining or ligating two or more antibody genes originally encoded for separate antibodies. For example, a chimeric antibody can be produced by combining or ligating two or more antibody genes (or fragments derived therefrom) from human, bovine, or murine species. In some embodiments, at least a portion of the antibody or antibody fragment can be of human or cynomolgus origin, but is not limited thereto. In certain embodiments, the antibodies described herein can be cross-reactive, e.g., the antibody can recognize human and cynomolgus antigens (e.g., human / canine antibodies).

[0077] "Antibody fragment" means an antibody in any form other than the full-length form. Antibody fragments herein include (i) antibodies that are small portions contained within a full-length antibody and (ii) artificially produced antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab and (Fab’)2, single-chain Fv (scFv), diabody, triabody, tetrabody, bifunctional hybrid antibody, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules having other scaffolds, bispecific antibodies, etc. (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Unless otherwise specified, the term "antibody (singular or plural)" used in the specification and claims may specifically include "antibody fragment (singular or plural)". In certain embodiments, "anti-CD3 antibody", "anti-CD3 Fab", "anti-CD3 Fab antibody", and "anti-CD3 Fab variant" are antibody fragments as defined herein.

[0078] As used herein, "computer-aided screening method" means any method for designing one or more mutations in a protein. This method utilizes a computer to evaluate the interaction energy between possible amino acid side-chain substitutions and / or the interaction energy between possible amino acid side-chain substitutions and the remaining portion of the protein.

[0079] As used herein, "full-length antibody" means the structure that constitutes the natural biological form of the H chain and / or L chain of an antibody. In many mammals (e.g., humans and mice), the above form is a tetramer, consisting of two pairs of combinations of two immunoglobulin chains (two pairs of the same combination). Each combination contains one light chain and one heavy chain. Each light chain contains the V L domain and C LIt contains a domain. Each heavy chain contains the V domain of immunoglobulin, the Cγ1 domain, the Cγ2 domain, and the Cγ3 domain. In each combination, the light chain variable region and the heavy chain variable regions (V and V) are jointly responsible for binding to the antigen. The constant regions (CL, Cγ1, Cγ2, and Cγ3, especially Cγ2 and Cγ3) are responsible for the effector functions of the antibody. In certain mammals (e.g., camels and llamas), the full-length antibody may consist of only two heavy chains. Each heavy chain contains the V domain of immunoglobulin, the Cγ2 domain, and the Cγ3 domain. H It contains the V domain of immunoglobulin, the Cγ1 domain, the Cγ2 domain, and the Cγ3 domain. In each combination, the light chain variable region and the heavy chain variable regions (V and V) are jointly responsible for binding to the antigen. The constant regions (CL, Cγ1, Cγ2, and Cγ3, especially Cγ2 and Cγ3) are responsible for the effector functions of the antibody. In certain mammals (e.g., camels and llamas), the full-length antibody may consist of only two heavy chains. Each heavy chain contains the V domain of immunoglobulin, the Cγ2 domain, and the Cγ3 domain. L and V H ) H It contains the V domain of immunoglobulin, the Cγ2 domain, and the Cγ3 domain.

[0080] As used herein, "immunoglobulin (Ig)" means a protein composed of one or more polypeptides substantially encoded by immunoglobulin genes. Examples of immunoglobulins include, but are not limited to, antibodies. Immunoglobulins may take various structural forms. For example, but not limited to, these include full-length antibodies, antibody fragments, individual domains of immunoglobulins (e.g., but not limited to, V, Cγ1, Cγ2, Cγ3, V, and C). H , Cγ1, Cγ2, Cγ3, V L and C L )

[0081] As used herein, "domain of immunoglobulin (Ig)" means a protein domain composed of a polypeptide substantially encoded by an immunoglobulin gene. Examples of Ig domains include, but are not limited to, V, Cγ1, Cγ2, Cγ3, V, and C. H , Cγ1, Cγ2, Cγ3, V L and C L )

[0082] As used herein, the term "mutant protein sequence" refers to a protein sequence having one or more residues with different amino acid identities from other similar protein sequences. The above "similar protein sequence" may be a natural wild-type protein sequence or a mutant type different from the wild-type sequence. Generally, the original sequence is expressed as the "parent sequence". The parent sequence may be a wild-type sequence or a mutant sequence. For example, in a preferred embodiment of the present invention, a humanized parent sequence may be used and a mutant type may be prepared by subjecting it to computer analysis.

[0083] As used herein, the "variable region" of an antibody refers to one or more polypeptides composed of the V H domain of an immunoglobulin, the V L domain of an immunoglobulin, or the V H domain and V L domain of an immunoglobulin (including mutants). The "variable region" may refer to these polypeptides in an isolated state (as in an Fv fragment, scFv fragment, or the region in a larger antibody fragment), or may refer to the region contained in a full-length antibody (or a scaffold molecule other than an antibody).

[0084] With respect to the anti-CD3 Fab antibody of the present invention, the terms "antigen-specific" or "specifically binds" refer to an anti-CD3 antibody that binds to one or more of a predetermined antigen or epitopes of a predetermined binding target in a sample containing a mixture containing the antigen, but does not substantially recognize or bind to other molecules.

[0085] As used herein, the term "bispecific anti-CD3 antibody" or "multispecific anti-CD3 antibody" refers to an anti-CD3 antibody having two or more antigen-binding sites or binding target-binding sites. Here, the first binding site has an affinity for a first antigen or epitope, and the second binding site has a binding affinity for a second antigen or epitope (different from the first antigen or epitope). In some embodiments, the bispecific anti-CD3 antibody has a binding site that binds to CD3 and one or more binding sites having a binding affinity for another antigen or epitope.

[0086] As used herein, the term "epitope" refers to a site on an antigen or a site on a binding target that is recognized by an anti-CD3 Fab antibody. When the antigen includes a polypeptide, the epitope can be an amino acid sequence or shape formed linearly or three-dimensionally. Also, the epitope can be any position on any type of antigen where an anti-CD3 antibody binds to the antigen.

[0087] As used herein, "antigen-binding polypeptide" or "anti-CD3 Fab antibody" includes polypeptides and proteins that have at least the biological activity of specific binding to a specific binding target (such as an antigen). Also included are CD3 analogs, CD3 isoforms, CD3 mimetics, CD3 fragments, hybrid CD3 proteins, fusion proteins, oligomers and multimers, homologs, glycosylation variants, and mutant proteins of the above polypeptides and proteins, regardless of the presence or absence of similar biological activity. Furthermore, the synthesis method or production method of these substances is not limited. Such synthesis or production methods include recombinant (regardless of whether it is prepared from cDNA, genomic DNA, synthetic DNA, or any other form of nucleic acid), methods by microinjection of nucleic acid molecules in vitro or in vivo, synthetic methods, transgenic methods, and gene activated methods (but not limited thereto). Specific examples of anti-CD3 antibodies include antibody molecules, heavy chains, light chains, variable regions, CDRs, Fabs, scFvs, non-antibody molecules having other frameworks, ligands, receptors, peptides, or any amino acid sequence that binds to an antigen (but not limited thereto).

[0088] Antigen-binding polypeptides include pharmaceutically acceptable salts and prodrugs. Prodrugs include salts, polymorphs, hydrates, solvates, fragments with biological activity, variants with biological activity, and stereoisomers of natural human anti-CD3 antibodies. Also included as the above prodrugs are salts, polymorphs, etc. of agonist variants, mimetics and antagonist variants of natural human anti-CD3 antibodies, and their polypeptide fusions. Also included within the term "antigen-binding polypeptide" are fusions having additional amino acids at the amino terminus, carboxyl terminus or both. Representative examples of fusions include, but are not limited to, methionyl anti-CD3 antibodies in which methionine is linked to the N-terminus of the anti-CD3 antibody as a result of recombinant expression. Also included are fusions for purification purposes (e.g., but not limited to, polyhistidine or affinity epitopes), fusions for linking an anti-CD3 antibody with another molecule having biological activity, fusions with serum albumin-binding peptides, and fusions with serum proteins (such as serum albumin), but not limited to these.

[0089] The term "antigen" or "binding target" refers to a substance that is the target of the binding activity exhibited by the anti-CD3 antibody. Substantially, any substance can be an antigen or binding target of the anti-CD3 Fab antibody.

[0090] Naturally produced antibodies (Abs) have a tetrameric structure consisting of two identical immunoglobulin (Ig) heavy chains and two identical immunoglobulin light chains. The heavy and light chains of Abs consist of different domains. Each light chain has one variable domain (VL) and one constant domain (CL). On the other hand, each heavy chain has one variable domain (VH) and three constant domains (CH). Each domain (consisting of approximately 110 amino acid residues) is folded into a characteristic β-sandwich structure formed by two antiparallel β-sheets (immunoglobulin fold). Each VL domain has three complementarity-determining regions (CDR1-3), and each VH domain has up to three complementarity-determining regions (CDR1-3). A CDR is a loop (or turn) that connects to a β-strand at one end of the variable domain. The variable regions of both the light and heavy chains are usually responsible for antigen specificity (although the contribution of each chain to specificity is not always equal). Antibody molecules have evolved to bind to a large number of molecules by randomizing the CDR loops.

[0091] The functional substructures of Abs can be created by proteolysis and recombinant methods. Abs contain Fab fragments, Fv fragments, and Fc portions. Fab fragments contain the VH-CH1 domains of the heavy chain and the VL-CL1 domains of the light chain, which are linked by a single interchain disulfide bond. Fv fragments contain only the VH and VL domains. The Fc portion contains the region of the antibody molecule that does not bind to the antigen. In some examples, even a single VH domain retains significant affinity for the antigen (Ward et al., Nature 341, 554-546, 1989). Also, certain monomeric κ light chains have been shown to specifically bind to their antigen (L. Masat et al., PNAS 91:893-896, 1994). It has been found that isolated light or heavy chains may also retain some antigen-binding activity (Ward et al., Nature 341, 554-546, 1989).

[0092] Among other functional substructures, there is a single-chain Fv (scFv). The scFv consists of the variable regions of the heavy and light chains of immunoglobulins, which are covalently linked to a peptide linker (S-z Hu et al., Cancer Research, 56, 3055-3061, 1996). These small proteins (Mr: 25,000 Da) usually remain in the state of a single polypeptide, retaining the specificity and affinity for antigens. And they can provide convenient building blocks for larger antigen-specific molecules. The scFv has a short circulatory half-life and, in many cases, its therapeutic usefulness is limited.

[0093] A small protein scaffold called "minibody" was designed using a part of the VH domain of Ig as a template (Pessi et al., Nature 362, 367-369, 1993). A minibody with high affinity for interleukin 6 (dissociation constant (K d ): about 10 -7 M) was discovered by randomizing the loops corresponding to CDR1 and CDR2 of VH and then selecting mutants by phage display method (Martin et al., EMBO J. 13, 5303-5309, 1994).

[0094] Analysis of IgG-like substances derived from camel serum usually shows the absence of the light chain variable domain. This suggests that sufficient specificity and affinity of the antibody can be derived from the VH domain (three or four CDR loops) only. "Camelized" VH domains with high affinity have been prepared. Also, high specificity can be obtained by randomizing only CDR3.

[0095] An alternative to the "minibody" is the "diabody". The diabody is a bivalent small antibody fragment with bispecificity and has two antigen-binding sites. This fragment contains the heavy chain variable domain (V H ) and the light chain variable domain (VL ) are included and they are linked on the same polypeptide chain (V H -V L ). The size of the diabody is comparable to that of the Fab fragment. The linker of the diabody is too short for the domains on the same chain to pair with each other. Therefore, each domain has to pair with the corresponding domain on the other chain. Thus, two antigen-binding sites are created. These dimeric antibody fragments (or "diabodies") are bivalent and bispecific (see P. Holliger et al., PNAS 90:6444-6448, 1993).

[0096] CDR peptides and CDR organic mimics have been prepared (Dougall et al., 1994, Trends Biotechnol. 12, 372-379). CDR peptides are short peptides, usually cyclic, corresponding to the amino acid sequences of the CDR loops of antibodies. The CDR loops are responsible for antibody-antigen interactions. CDR peptides and CDR organic mimics have been shown to retain some degree of binding affinity (Smyth & von Itzstein, J. Am. Chem. Soc. 116, 2725-2733, 1994). The CDRs of mice have been transplanted onto the human Ig backbone without loss of affinity (Jones et al., 1986, Nature 321, 522-525; Riechmann et al., 1988).

[0097] In the human body, specific Abs are selected and amplified from a vast library (affinity maturation). This process can be reproduced in vitro using combinatorial library technology. By successfully presenting Ab fragments on the surface of bacteriophages, it becomes possible to create and screen a large number of CDR variants (McCafferty et al., Nature 348, 552 - 554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88, 7978 - 7982, 1991; Winter et al., Annu. Rev. Immunol. 12, 433 - 455, 1994). The production of Fabs and Fvs (and their derivatives) generated by this technology is increasing. Combinatorial technology can also be combined with mimetic Abs.

[0098] Numerous protein domains that could potentially serve as protein scaffolds have been expressed as fusions to phage capsid proteins. A review of this is [Clackson & See [Wells, Trends Biotechnol. 12:173-184, 1994]. Some of these protein domains have already been used as scaffolds for presenting random peptide sequences. Examples of this include bovine pancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433, 1992), human growth hormone (Lowman et al., Biochemistry 30:10832-10838, 1991; Venturini et al., Protein Peptide Letters 1:70-75, 1994), and the IgG-binding domain of streptococcus (O'Neil et al., Techniques in Protein Chemistry V (Crabb, L,. ed.) pp. 517-524, Academic Press, San Diego, 1994). These scaffolds present one randomized loop or region. As a scaffold for presentation on the filamentous phage M13, tendamistat has been used (McConnell and Hoess, J. Mol. Biol. 250:460-470, 1995).

[0099] By a method of covalently attaching poly(ethylene glycol) (abbreviated PEG), a hydrophilic polymer, (i) water solubility and bioavailability are improved, (ii) serum half-life and therapeutic half-life are increased, (iii) immunogenicity and biological activity are regulated, or (iv) for molecules having many biological activities (e.g., proteins, peptides, and particularly hydrophobic molecules), the circulation time is extended. In pharmaceuticals, artificial implants, and other applications where biocompatibility, removal of toxicity and removal of immunogenicity are important, PEG has been widely used. To maximize the desired properties of PEG, the total molecular weight and the hydration state of the PEG polymer or the polymer added to the molecule having biological activity must be made sufficiently high. In this way, the general advantages associated with the addition of the PEG polymer can be imparted (such as increasing water solubility or extending the half-life in circulation without adversely affecting the biological activity of the parent molecule).

[0100] Often, PEG derivatives are linked to molecules having biological activity via reactive chemical functional sites (such as lysine residues, cysteine residues and histidine residues, N-terminus, and hydrocarbon moieties). Proteins and other molecules usually have only a limited number of reaction sites available for the addition of polymers. Usually, the sites most suitable for polymer addition modification play an important role in receptor binding and are essential for maintaining the biological activity of the molecule. As a result, adding polymer chains indiscriminately to the reaction sites of molecules having biological activity usually significantly reduces or completely loses the biological activity of the polymer-modified molecules (R. Clark et al., J. Biol. Chem., 271:21969-21977, 1996). The conventional approaches for forming a complex having a polymer molecular weight sufficient to impart the desired advantages to the target molecule generally relate to randomly adding a large number of polymer chains to the molecule. Therefore, it was an approach with a high risk of reducing or completely losing the biological activity of the parent molecule.

[0101] The reaction sites that form loci for the addition of PEG derivatives to proteins are specified by the protein's structure. Proteins, including enzymes, are composed of various sequences of α-amino acids having the general structure H2N--CHR--COOH. The α-amino portion (H2N--) of one amino acid binds to the carboxyl portion (--COOH) of the adjacent amino acid to form an amide bond, which can be represented as --(NH--CHR--CO) n --n, where the subscript "n" can be equivalent to hundreds or thousands. The fragment represented by R can have a reaction site for protein bioactivity and the addition of PEG derivatives.

[0102] For example, in the amino acid lysine, --NH2 moieties are present at the ε-position and the α-position. ε--NH2 is free for reaction under basic pH conditions. Many techniques in the field of protein derivatization with PEG are related to the development of PEG derivatives for the addition to the ε--NH2 moiety of lysine residues present in proteins ("Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, pp. 1-17, 2003). However, all of these PEG derivatives have a general limitation that they cannot be selectively installed among the numerous lysine residues present on the surface of the protein. This can be a significant limitation when lysine residues are involved in mediating the interaction of the protein with other biological molecules, such as when the lysine residue is important for protein activity, for example, when present in the enzyme active site, or in the case of the receptor binding site.

[0103] Similar to the first serious problem with existing methods of PEGylating proteins, a second serious problem is that PEG derivatives can undergo unwanted side reactions with residues other than the desired ones. Histidine has a reactive imino moiety that is structurally represented as --N(H)--, but many chemically reactive species that react with ε--NH2 can also react with --N(H)--. Similarly, the side chain of the amino acid cysteine has a free sulfhydryl group that is structurally represented as -SH. In some cases, PEG derivatives directed at the ε--NH2 group of lysine also react with cysteine, histidine, or other residues. This creates a complex and heterogeneous mixture of PEGylated bioactive molecules and risks destroying the activity of the targeted bioactive molecule. It is desirable to develop PEG derivatives that allow the introduction of chemical functionality at one site within a protein, resulting in the selective coupling of one or more PEG polymers to a bioactive molecule at specific, defined, and predictable sites on the protein surface.

[0104] In addition to lysine residues, considerable effort has been directed in the art towards the development of activated PEG reagents that target the side chains of other amino acids, including cysteine, histidine, and the N-terminus. For example, U.S. Patent No. 6,610,281, and ["Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, pp. See [1-17, 2003] (incorporated herein by reference). Cysteine residues can be introduced site-selectively into the structure of a protein using site-directed mutagenesis and other techniques known in the art, and the resulting free sulfhydryl moieties can be reacted with PEG derivatives having thiol-reactive functional groups. However, this approach has the drawback that the introduction of free sulfhydryl groups can complicate the expression, folding, and stability of the resulting protein. Thus, there is a need for means of introducing chemical functional groups into bioactive molecules that allow for the selective coupling of one or more PEG polymers to a protein while being compatible with (i.e., not causing undesirable side reactions with) sulfhydryl and other chemical functional groups typically found in proteins.

[0105] As is recognized in the art, many of these derivatives developed for addition to the side chains of proteins, particularly the --NH2 moiety on the side chain of lysine amino acids and the -SH moiety on cysteine side chains, have proven to have problems in their synthesis and use. Some degrade by hydrolysis or form unstable bonds with proteins that are unstable in an aqueous environment such as in the bloodstream. Some form more stable bonds but are hydrolyzed before the bond is formed, meaning that the reactive groups on the PEG derivative can be inactivated prior to addition to the protein. Some are somewhat toxic and thus not well suited for in vivo use. Some are too slow reacting to be practical. Some cause loss of protein activity by being added to sites involved in protein activity. Some are not specific for the site to which they are added, which can also result in loss of the desired activity and lack of reproducibility of results. To overcome the problems associated with modifying proteins having poly(ethylene glycol) moieties, more stable PEG derivatives (e.g., U.S. Patent No. 6,602,498, incorporated herein by reference), or PEG derivatives that selectively react with thiol moieties on molecules and surfaces (e.g., U.S. Patent No. 6,610,281, incorporated herein by reference) have been developed. There is clearly a need in the art for PEG derivatives that are chemically inert in a physiological environment until required to react selectively to form stable chemical bonds.

[0106] Recently, entirely new technologies in protein science have been reported, which have the potential to overcome many of the limitations associated with site-specific modification of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (e.g., [L. Wang, et al, Science 292:498-500, 2001]) and the eukaryote Saccharomyces cerevisiae (S. cerevisiae) (e.g., [J. Chin et al, Science 301:964-7, 2003]). This has enabled the in vivo incorporation of genetically non-encoded amino acids into proteins. Using this method, numerous new amino acids with novel chemical, physical, or biological properties (such as photoaffinity labels and photo-isomerizable amino acids, keto amino acids, and glycosylated amino acids) have been efficiently and with high accuracy incorporated into proteins in E. coli and yeast in response to the amber codon TAG. See, for example, [J. W. Chin et al, Journal of the American Chemical Society 124:9026-9027, 2002], [J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137], [J. W. Chin, et al, PNAS United States of America 99:11020-11024, 2002], and [L. Wang, & P. G. Schultz, Chem. Comm., 1-10, 2002]. These studies have demonstrated that chemical functional groups (such as ketone groups, alkyne groups, and azide moieties) not found in proteins, which are chemically inert to all of the functional groups found in the amino acids encoded by the normal 20 genes and can be used to react efficiently and selectively to form stable covalent bonds, can be selectively and routinely introduced into proteins.

[0107] Non-genetically encoded amino acids can be incorporated into proteins. This allows the introduction of chemical functional groups that can provide various alternatives to natural functional groups (e.g., the ε-NH2 of lysine, the sulfhydryl -SH of cysteine, the imino group of histidine, etc.). Certain chemical functional groups are known to be inert towards the functional groups of the normal 20 amino acids (amino acids encoded by genes), while reacting neatly and efficiently to form stable bonds. For example, azide groups and acetylene groups are known to undergo the Huisgen [3 + 2] cycloaddition reaction in aqueous conditions and in the presence of a catalytic amount of copper (see, for example, Tornoe, et al., (2002) Org. Chem. 67:3057 - 3064; Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596 - 2599). By introducing an azide moiety into the protein structure, functional groups can be incorporated that are chemically inert towards, for example, amine groups, sulfhydryl groups, carboxylic acid groups, and hydroxyl groups in the protein, while reacting smoothly and efficiently with an acetylene moiety to form a cycloaddition product. Importantly, in the absence of an acetylene moiety, the azide remains chemically inert and does not react in the presence of other protein side chains and under physiological conditions.

[0108] Various documents disclose the modification of polypeptides by complexation with polymers or glycosylation. The terms "anti-CD3 antibody" or "antigen-binding polypeptide" refer to polypeptides that retain one or more of the biological activities of natural antibodies in addition to the anti-CD3 antibodies described above. Such biological activities include (but are not limited to) activities other than binding to an antigen. Activities other than binding to an antigen include (but are not limited to) any one or more activities associated with Fc. The terms "anti-CD3 antibody" or "antigen-binding polypeptide" include polypeptides complexed with a polymer such as PEG, wherein one or more of cysteine, lysine, N-terminal or C-terminal amino acids, or other residues may be additionally derivatized (but are not limited to these). Furthermore, the anti-CD3 antibody may have a linker, a polymer or a molecule having biological activity. Here, the amino acids complexed with the linker, polymer or molecule having biological activity may be non-natural amino acids according to the present invention. Alternatively, known techniques (such as coupling with lysine or cysteine) may be used to complex with naturally encoded amino acids. U.S. Patent No. 4,904,584 discloses a PEGylated polypeptide with reduced lysine. The PEGylated polypeptide has one or more lysine residues deleted or substituted with other amino acid residues. International Publication No. 99 / 67291 discloses a method for complexing a protein with PEG. In the above method, one or more amino acid residues of the protein are deleted, and the protein is contacted with PEG under conditions sufficient for complexation of the protein. International Publication No. 99 / 03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily. The cysteine residues of the PEGylated variants are substituted with non-essential amino acid residues located in specific regions of the polypeptides. International Publication No. 00 / 26354 discloses a method for producing a glycosylated polypeptide variant with reduced allergenicity. The polypeptide variant has one or more additional glycosylation sites compared to the corresponding parent polypeptide.

[0109] The term "antigen-binding polypeptide" also includes glycosylated anti-CD3 antibodies. A glycosylated anti-CD3 antibody is a polypeptide glycosylated at any amino acid position, such as one that is N-linked or O-linked glycosylated (but not limited thereto). Also, a variant having a single nucleotide mutation is considered an anti-CD3 antibody variant having biological activity. Furthermore, splicing variants are also included in the antigen-binding polypeptide. The term "antigen-binding polypeptide" also includes heterodimers, homodimers, heteromultimers or homomultimers of one or more arbitrary anti-CD3 antibodies. Alternatively, the antigen-binding polypeptide includes substances that are chemically linked to any other polypeptide, protein, hydrocarbon, polymer, small molecule, linker, ligand, or any other type of molecule having biological activity, or are expressed as a fusion protein. Similarly, for example, polypeptide analogs having specific deletions or other modifications and still maintaining biological activity are also included.

[0110] In some embodiments, the antigen-binding polypeptide further has an addition, substitution or deletion that modulates the biological activity of the anti-CD3 antibody. For example, one or more of the properties or activities of the anti-CD3 antibody may be modulated by the addition, substitution or deletion. Examples of such modulation include: (i) modulation of the affinity for the antigen; (ii) modulation of the change in the conformation or other secondary, tertiary, or quaternary structure of the antigen (e.g., increase or decrease in the amount of change, but not limited thereto); (iii) stabilization of the change in the conformation or other secondary, tertiary, or quaternary structure of the antigen; (iv) induction of the change in the conformation or other secondary, tertiary, or quaternary structure of the antigen; (v) modulation of the circulation half-life; (vi) modulation of the therapeutic half-life; (vii) modulation of the stability of the polypeptide; (viii) modulation of the dosage; (ix) modulation of the release or bioavailability; (x) facilitation of purification; (xi) improvement or alteration of a specific route of administration (however, not limited thereto). Similarly, the antigen-binding polypeptide may have a protease cleavage sequence, a reactive group, an antibody-binding domain (e.g., FLAG or poly-His, but not limited thereto), or other affinity sequences (e.g., FLAG, poly-His, GST, etc., but not limited thereto), or a binding molecule (e.g., biotin, but not limited thereto). The above binding molecules include those that facilitate the detection of the polypeptide (e.g., GFP, but not limited thereto), those that facilitate purification, and those that improve other properties.

[0111] The term "antigen-binding polypeptide" also includes homodimers, heterodimers, homotrimers and heterotrimers of the linked anti-CD3 antibodies. Examples of the linking mode include, but are not limited to, (i) direct linking via side chains of non-naturally encoded amino acids, (ii) linking between side chains of the same or different non-naturally encoded amino acids, (iii) linking between side chains of naturally encoded amino acids, (iv) linking by fusion, or (v) indirect linking via a linker. Representative examples of the linker include, but are not limited to, small organic compounds, water-soluble polymers of various lengths (such as poly(ethylene glycol), polydextran or polypeptide of various lengths).

[0112] Those skilled in the art will understand that the position of an amino acid corresponding to a position in a specific antigen-binding polypeptide sequence can be easily identified in a fragment of the antigen-binding polypeptide or a related antigen-binding polypeptide, etc. For example, using a sequence alignment program (such as BLAST), the specific position in a protein corresponding to a position in a related sequence can be aligned and identified.

[0113] The term "antigen-binding polypeptide" also includes antigen-binding polypeptides having one or more amino acid substitutions, additions or deletions. The antigen-binding polypeptide of the present invention may have a modification in which one or more natural amino acids are complexed with one or more non-natural amino acids. Representative examples of substitutions at various amino acid positions in the natural anti-CD3 antibody polypeptide have been described above. For example, substitutions that modulate one or more of the biological activities of the antigen-binding polypeptide can be mentioned (but are not limited to these). Specifically, it includes improvement of agonist activity, improvement of polypeptide solubility, conversion of the polypeptide into an antagonist, etc. (but is not limited to these). These examples are also included in the term "anti-CD3 antibody".

[0114] "Non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrrolysine or selenocysteine. Terms that can be used as synonyms for the term "non-naturally encoded amino acid" include "non-natural amino acid, unnatural amino acid (「non-natural amino acid」, 「unnatural amino acid」, 「non-naturally-occurring amino acid」, 「non-canonical amino acid」)", and forms with or without hyphens attached to the above terms. The term "non-naturally encoded amino acid" includes amino acids that are produced by modification (e.g., post-translational modification) of naturally encoded amino acids (such as the 20 common amino acids or pyrrolysine and selenocysteine, but not limited to these), but that are not themselves typically incorporated into the polypeptide chain extended by the translation complex (however, not limited to these). Examples of such non-natural amino acids include N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine (however, not limited to these). In some embodiments, the non-natural amino acid has a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids also include those in which the natural N- or O-bond between the amino acid and the sugar has been replaced by a covalent bond not commonly found in nature. Such amino acids include, but are not limited to, alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include sugars (2-deoxy-glucose, 2-deoxygalactose, etc.) not commonly found in natural proteins.Specific examples of non-natural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propargyloxy phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine.

[0115] The term "amino-terminal modifying group" refers to any molecule that can be added to the amino terminus of a polypeptide. Similarly, the term "carboxy-terminal modifying group" refers to any molecule that can be added to the carboxy terminus of a polypeptide. Examples of terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins (such as serum albumin), or other moieties that extend the serum half-life of a peptide.

[0116] The terms "functional group", "active site", "activating group", "leaving group", "reaction site", "chemical reaction group", and "chemical reaction site" are used in the art and in this specification to denote distinguishable and definable moieties or units within a molecule. These terms generally have the same meaning in the art of chemistry. Further, in this specification, they are used to denote portions of a molecule that have some function or activity or that react with other molecules.

[0117] As used herein, the term "linkage" or "linker" is used to denote a group or bond that is typically formed as a result of a chemical reaction and is typically a covalent bond. A "bond stable to hydrolysis" means that the bond is substantially stable in water and does not react with water at practical pH values (e.g., it does not react under physiological conditions for an extended period of time, and in some cases, indefinitely). A "bond unstable to hydrolysis" or "hydrolyzable bond" means that the bond is degradable in water or an aqueous solution (e.g., in blood). A "bond unstable to enzymes" or "enzymatically degradable bond" means that the bond can be degraded by one or more enzymes. As is known in the art, PEG and related polymers may have a degradable bond (i) in the polymer backbone or (ii) in a linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. For example, an ester bond formed by the reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group of a bioactive agent typically hydrolyzes under physiological conditions to release the agent. Other hydrolyzable bonds include (i) a carbonate bond, (ii) an imine bond resulting from the reaction of an amine and an aldehyde, (iii) a phosphate ester bond formed by the reaction of an alcohol and a phosphate group, (iv) a hydrazone bond that is a reaction product of a hydrazide and an aldehyde, (v) an acetal bond that is a reaction product of an aldehyde and an alcohol, (vi) an orthoester bond that is a reaction product of a formate and an alcohol, (vii) a peptide bond formed by an amine group (e.g., but not limited to, at the end of a polymer such as PEG) and a carboxy group of a peptide, (viii) an oligonucleotide bond formed by a phosphoramidite group (e.g., but not limited to, at the end of a polymer) and a 5'-hydroxyl group of an oligonucleotide (however, it is not limited thereto). In the antigen-binding polypeptide of the present invention, a branched linker may be used.

[0118] As used herein, the terms "bioactive molecule", "bioactive moiety", or "bioactive agent" mean any substance that can affect any physical or biochemical property of an interaction related to a biological system, biological pathway, biological molecule, or organism (e.g., but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans). Specifically, as used herein, "bioactive molecule" includes (i) any substance for the purpose of diagnosing, curing, alleviating, treating, or preventing diseases in humans or other animals, or (ii) any substance for the purpose of improving the physical or mental well-being of humans or animals (but not limited thereto). Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Types of bioactive agents preferably used in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, contrast agents, polymers, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroid agents, toxins derived from microorganisms, and the like.

[0119] The anti-CD3Fab-folate antibody of the present invention may be complexed with a molecule such as PEG to improve delivery and pharmacokinetic profiles in vivo. Leong et al. disclosed that site-specific PEGylation of the Fab' fragment of an anti-IL-8 antibody reduced the clearance rate compared to the non-PEGylated form with little (or no) loss of antigen-binding activity (Leong, S.R. et al. (2001) Cytokine 16:106-119).

[0120] In addition, numerous cleavable linkers are known to those skilled in the art (see U.S. Patent Nos. 4,618,492, 4,542,225, and 4,625,014). Examples of mechanisms by which drugs are released from these linker groups include irradiation of light-sensitive bonds with light and acid-catalyzed hydrolysis. For example, U.S. Patent No. 4,671,958 describes an immunocomplex having a linker that is cleaved at the target site by a proteolytic enzyme of the patient's complement system in vivo. The length of the linker may be predetermined or may be selected according to the desired spatial relationship between the anti-CD3 antibody and the molecule linked thereto. Numerous reports have been made on methods for attaching various radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies. From this perspective, those skilled in the art would be able to determine an appropriate method for attaching a given agent to an anti-CD3 antibody or other polypeptide.

[0121] "Bifunctional polymer" refers to a polymer having two separate functional groups. These functional groups can specifically react with other moieties (such as, but not limited to, the side chains of amino acids) to form covalent or non-covalent bonds. A bifunctional linker having (i) one functional group capable of reacting with a group of a component having a specific biological activity and (ii) the other group capable of reacting with a group of a second biological component may be used for the formation of a complex comprising a component having a first biological activity, the bifunctional linker and a component having a second biological activity. Many methods and linker molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 188,256, U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, 4,569,789 and 4,589,071 (incorporated herein by reference). "Multifunctional polymer" refers to a polymer having two or more separate functional groups. These functional groups can specifically react with other moieties (such as, but not limited to, the side chains of amino acids) to form covalent or non-covalent bonds. The bifunctional polymer or multifunctional polymer may have an arbitrary and desired molecular length or molecular weight. Also, it may be selected to provide a desired and specific gap or configuration between the molecules linked to the anti-CD3 antibody.

[0122] As used herein, the term "water-soluble polymer" refers to any polymer that can be dissolved in an aqueous solvent. When a water-soluble polymer binds to an anti-CD3 antibody, for example, the following changes may occur (but are not limited to): (i) an extension or regulation of the serum half-life or the therapeutic half-life as compared to the unmodified one, (ii) a regulation of immunogenicity, (iii) a regulation of physical association properties (such as aggregation and multimer formation), (iv) a modification of the binding to a receptor, and a modification of the dimerization or multimerization of the receptor. The water-soluble polymer may or may not have biological activity per se. Also, the water-soluble polymer may be used as a linker for adding other substances to the anti-CD3 antibody. Examples of other substances include, but are not limited to, one or more anti-CD3 antibodies or one or more molecules having biological activity. Suitable polymers include, but are not limited to, the following: polyethylene glycol, polyethylene glycol-propionaldehyde, mono C1-C10 alkoxy derivatives or aryloxy derivatives of polyethylene glycol (described in U.S. Patent No. 5,252,714, incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether-maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives (such as dextran sulfate), polypropylene glycol, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (such as, but not limited to, methylcellulose and carboxymethylcellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, polyalkylene glycol copolymers and their derivatives, polyvinyl ethyl ether, α-β-poly[(2-hydroxyethyl)-DL-asparagine amide, etc., or mixtures thereof.Examples of such water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

[0123] As used herein, the terms "polyalkylene glycol" or "poly(alkylene glycol)" refer to polyethylene glycol (poly(ethylene glycol)), polypropylene glycol, polybutylene glycol, and derivatives thereof. The term "polyalkylene glycol" includes both linear and branched polymers. The average molecular weight of the polyalkylene glycol is from 0.1 kDa to 100 kDa. In some embodiments, the "polyalkylene glycol" or "poly(alkylene glycol)" may be in the range of about 5K to 50K, or 5K to 50K. For example, other exemplary embodiments are summarized in the catalogs of commercial manufacturers (such as the catalog of Shearwater Corporation, "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001)). As used herein, poly(ethylene glycol) having a molecular weight such as 5 kDa, 10 kDa, 20 kDa, etc. are represented as "5K PEG", "10K PEG", "20K PEG", etc., respectively.

[0124] As used herein, the term "regulated serum half-life / regulating serum half-life" means a positive or negative change in the circulating half-life when a modified anti-CD3 antibody is compared to an unmodified one. Serum half-life is measured by taking blood samples at various time points after administration of the anti-CD3 antibody and determining the concentration of the molecule in each sample. Since there is a correlation between serum concentration and time, the serum half-life can be calculated. An extension of the serum half-life by about two-fold or more is desirable, although in some cases, such as providing a sufficient dosing schedule or preventing toxic effects, a smaller extension of the serum half-life may also be useful. In some embodiments, the extension of the serum half-life is about three-fold or more, about five-fold or more, about ten-fold or more, about fifteen-fold or more, about twenty-fold or more, about twenty-five-fold or more, about thirty-fold or more, about forty-fold or more, or about fifty-fold or more.

[0125] As used herein, the term "regulated therapeutic half-life / regulating therapeutic half-life" means a positive or negative change in the half-life when a therapeutically effective amount of (i) an anti-CD3 antibody, or (ii) an anti-CD3 antibody having a modified bioactive molecule is compared to an unmodified one. Therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. It is desirable to extend the therapeutic half-life to provide certain advantages in the dosing schedule or total dose administered, or to avoid undesirable effects. In some embodiments, the therapeutic half-life is extended by enhancing efficacy, strengthening or weakening the binding of the modified molecule to its target, or increasing or decreasing other parameters or mechanisms of action of the unmodified molecule.

[0126] As used herein, the term "isolated / isolation" when used in reference to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components that are associated with it in its natural state. This can be in a homogeneous state. An isolated substance can be in a dry state, a semi-dry state, or a solution state (e.g., but not limited to, an aqueous solution). Typically, techniques of analytical science (such as polyacrylamide gel electrophoresis or high performance liquid chromatography) are used to determine purity and homogeneity. A protein that predominates among the types present in a preparation is substantially purified. Specifically, an isolated gene is separated from the open reading frames that are located adjacent to the gene and that encode proteins other than those of the gene. As used herein, the term "purified / purification" means that a nucleic acid or protein migrates as substantially a single band in an electrophoretic gel. Specifically, "purified" means that the nucleic acid or protein has a purity of 85% or greater, 90% or greater, 95% or greater, 99% or greater, or more.

[0127] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof. "Nucleic acid" may be in single-stranded or double-stranded form. Unless specifically limited, this term also includes nucleic acids containing known analogs of natural nucleotides. This analog has binding properties equivalent to those of the nucleic acid being compared and is metabolized in the same manner as naturally occurring nucleotides. Unless specifically limited otherwise, this term also refers to oligonucleotide analogs (such as PNA (peptide nucleic acid)), DNA analogs used in antisense technology (such as phosphorothioate, phosphoramidate, etc.). Unless otherwise stated, a particular nucleic acid sequence, in addition to the explicitly shown sequence, also substantially includes (i) conservatively modified variants (e.g., but not limited to, substitutions by degenerate codons), and (ii) complementary sequences. Specifically, substitution by degenerate codons may be performed by generating a sequence in which the third position of one or more (or all) selected codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0128] In this specification, the terms "polypeptide", "peptide" and "protein" are used interchangeably to represent polymers of amino acid residues. That is, descriptions regarding polypeptides are equally applicable to descriptions regarding peptides and proteins (and vice versa). This term is applicable to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are amino acids that are not naturally encoded. As used herein, this term includes amino acid chains of any length (for example, full-length proteins (i.e., antigens) in which amino acid residues are linked by covalent peptide bonds).

[0129] The term "amino acid" refers to naturally occurring amino acids and non-naturally occurring amino acids, and in addition thereto, amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), as well as pyrrolysine and selenocysteine. "Amino acid analog" refers to a compound having the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to hydrogen, a carboxy group, an amino group and an R group) (such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, etc.). Such analogs have a modified R group (such as norleucine, etc.) or a modified peptide backbone. However, the same basic chemical structure as a naturally occurring amino acid is preserved.

[0130] In this specification, amino acids may be represented by the commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by the commonly used one-letter symbols.

[0131] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, a "conservatively modified variant" refers to a nucleic acid encoding the same or essentially the same amino acid sequence. Where the nucleic acid does not encode an amino acid sequence, it refers to an essentially identical sequence. Due to the degeneracy of the genetic code, there are numerous functionally identical nucleic acids encoding any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, anywhere an alanine is specified by a codon, the codon can be changed to any of the corresponding codons above without changing the encoded polypeptide. Such nucleic acid mutations are "silent mutations" and are a type of conservatively modified variant. It is assumed that all possible silent mutations of such nucleic acids are described by all nucleic acid sequences encoding the polypeptide herein. One of ordinary skill in the art will understand that each codon in a nucleic acid can be modified to obtain a functionally identical molecule (usually excluding the AUG, which is the only codon for methionine, and the TGG, which is the only codon for tryptophan). Thus, each silent mutation of a nucleic acid encoding a polypeptide is substantially included in each described sequence.

[0132] With respect to an amino acid sequence, one of ordinary skill in the art will understand that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence resulting in the change, addition, or deletion of one or a small percentage of amino acids in the encoded sequence are also "conservatively modified variants" (if, as a result of the change, an amino acid is substituted with an amino acid that is chemically similar). Lists of conservative substitutions providing functionally similar amino acids are well known in the art. Furthermore, such conservatively modified variants include polymorphic variants, interspecies homologs, and alleles of the present invention.

[0133] The following eight groups each describe amino acids that are conservatively substituted for one another: (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine (C), methionine (M) (see, e.g., [Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)]).

[0134] The terms "identical" or "identity" percent, when referring to two or more nucleic acid or two or more polypeptide sequences, refer to two or more identical sequences or two or more identical subsequences. When compared and aligned to maximize correspondence over a comparison window or specified region using one of the following sequence comparison algorithms or by manual alignment and visual inspection, the sequences are "substantially identical" if they have a percentage of amino acid residues or nucleotides that are identical (i.e., about 60% identity over a particular region, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity). This definition also refers to the complement of the test sequence. Identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, if not specified, over the entire sequence or entire polynucleotide or entire polypeptide.

[0135] When comparing arrays, typically one array serves as the reference array against which the test array is compared. When using an array comparison algorithm, the test array and the reference array are input into a computer, and if necessary, subarray coordinates are specified and array algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. Then, the array comparison algorithm calculates the percent sequence identity of the test array relative to the reference array based on the program parameters.

[0136] As used herein, the term "subject" refers to an animal that is the object of treatment, observation, or experiment, preferably a mammal, and most preferably a human.

[0137] As used herein, the term "effective amount / effective quantity" means that one or more of the symptoms of a disease, condition, or disorder to be treated are reduced to some extent by the dosage of the (modified) unnatural amino acid polypeptide. Compositions containing the (modified) unnatural amino acid polypeptides described herein can be administered for prophylactic treatment, enhancing treatment, and / or therapeutic treatment.

[0138] The term "enhance / enhancing" means to increase or extend a desired effect in terms of either strength or duration. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhance / enhancing" refers to the ability to increase or extend the effect of another therapeutic agent in a system in terms of either strength or duration. As used herein, "an amount effective for enhancement" means an amount sufficient to enhance the effect of a therapeutic agent in a desired system. When used with respect to a patient, an amount effective for such use depends on the severity and course of the disease, disorder, or condition, the patient's treatment history, the patient's health status and response to the drug, and the judgment of the treating physician.

[0139] As used herein, the term "modified / modifed" refers to the presence of post-translational modifications in a polypeptide. The form of the term "(modified)" means that the polypeptide under discussion is optionally modified. That is, the polypeptide under discussion may or may not be modified.

[0140] The terms "post-translationally modified" and "modified" refer to any modification of a natural or non-natural amino acid that occurs after the amino acid has been incorporated into the polypeptide chain. This term includes in vivo modifications during translation, in vivo modifications after translation, and in vitro modifications after translation (however, the above are merely examples).

[0141] In prophylactic or therapeutic applications, a composition comprising a (modified) non-natural amino acid polypeptide is administered to a patient already suffering from a disease, condition or disorder in an amount sufficient for treatment (or an amount that at least partially suppresses the symptoms of the disease, disorder or condition). Such an amount is defined as a "prophylactically effective amount" or a "therapeutically effective amount". The prophylactically effective amount or therapeutically effective amount depends on the severity and course of the disease, disorder or condition, the treatment history, the health status of the patient and the response to the drug, as well as the judgment of the treating physician. It is understood in the art that such a therapeutically effective amount can be determined by routine experimentation (e.g., dose escalation clinical trials).

[0142] The term "treatment" is used to represent either prophylactic treatment and / or therapeutic treatment.

[0143] Unless otherwise stated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacy within the scope of the art are employed.

[0144] [Detailed Description of the Invention] [Introduction] The inventors have developed bispecific antibodies comprising a molecule or agent having biological activity in the absence or presence of a water-soluble polymer molecule. In certain embodiments, the present invention provides a bispecific antibody comprising an anti-CD3 antibody, fragment or variant comprising one or more folic acid molecules in the absence or presence of one or more PEG molecules. The one or more PEG molecules may be single PEG or double PEG, for example, single or double 5K PEG, 10K PEG, 20K PEG or more. The one or more PEG molecules can be linear or branched. The anti-CD3 antibody, fragment or variant comprises an anti-CD3 Fab antibody engineered to have one or more non-naturally encoded amino acids (such as para-acetylphenylalanine (pAF), etc.) at any suitable position (heavy chain at positions 114, 115, 129 or 160 (Kabat numbering), and light chain at positions 157, 172, 205 (Kabat numbering), including but not limited to these) of the Fab heavy or light chain amino acid sequence. The bispecific antibody may comprise an anti-CD3 Fab engineered to have one or more non-naturally encoded amino acids (such as para-acetylphenylalanine (pAF)) on the heavy chain (K129; Kabat numbering) and light chain (L157; Kabat numbering) of the Fab. The bispecific antibody may consist of an anti-CD3 Fab engineered to have one or more non-naturally encoded amino acids (such as para-acetylphenylalanine (pAF)) on the heavy chain (K129; Kabat numbering) and light chain (L157; Kabat numbering) of the Fab. In some embodiments of the present invention, the one or more PEG molecules can be complexed or linked to one or more folic acid molecules complexed to one or more non-natural amino acids (such as pAF), and incorporated into the anti-CD3 antibody using unique oxime chemistry, resulting in, for example, one or two PEG molecules and / or folic acid molecules stably complexed with the anti-CD3 Fab. By adding one or more PEG molecules (such as 5K PEG, 10K PEG, or 20K PEG), the pharmacokinetic properties of the bispecific antibody are significantly improved while maintaining specific cytotoxicity against FOLR1-expressing cells both in vitro and in vivo.Pre-tumorigenic macrophages (M2) and MDSC cells were observed to be preferentially reduced by the PEGylated anti-CD3Fab-folate compositions described herein. These results suggest that by improving the pharmacokinetic properties, the number of administrations can be reduced and administration using an infusion pump can be avoided. As used herein, the terms "PEG-folate" or "folate-PEG" and the terms "BiPEG-BiFolate" or "BiFolate-BiPEG" are used interchangeably.

[0145] [Antibodies, antibody fragments and variants thereof] The antibody, antibody fragment, or variant of the present invention can be a human antibody or antibody fragment, a humanized antibody or antibody fragment, an engineered antibody or antibody fragment, a non-human antibody or antibody fragment, and / or a chimeric antibody or antibody fragment. The antibody, antibody fragment, or variant herein can have two or more amino acid sequences. The first amino acid sequence can include a first antibody chain, and the second amino acid sequence can include a second antibody chain. The first antibody chain can have a first amino acid sequence, and the second antibody chain can have a second amino acid sequence. The chains of an antibody can refer to an antibody heavy chain, an antibody light chain, or a combination of regions, or all of the antibody heavy chain and regions, or all of the antibody light chain. As a non-limiting example, the antibody herein includes a heavy chain, fragment, or variant thereof, and a light chain, fragment, or variant thereof. The two amino acid sequences of the antibody (including two antibody chains) can be linked by one or more disulfide bonds, chemical linkers, peptide linkers, or combinations thereof. A chemical linker includes a linker via non-natural amino acids. A chemical linker includes a linker via one or more non-natural amino acids. A chemical linker can include a chemical conjugate. A peptide linker has any amino acid sequence that links two amino acid sequences. A peptide linker can include 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. A peptide linker can be a part of any antibody, including domains of an antibody such as a variable domain, CH1, CH2, CH3, and / or CL domain. In some embodiments, the heavy chain and the light chain are linked via, for example, a peptide linker. In some examples, the heavy chain and the light chain are linked by, for example, one or more disulfide bonds.

[0146] The antibodies, antibody fragments, and antibody variants described in the present invention can interact or engage with antigens on effector cells. Effector cells include, but are not limited to, immune cells, genetically modified cells with altered cytotoxic activity, cells related to host defense mechanisms, anti-inflammatory cells, white blood cells, lymphocytes, macrophages, red blood cells, platelets, neutrophils, monocytes, eosinophils, basophils, mast cells, NK cells, B cells, or T cells. In some embodiments, the immune cell can be a T cell (cytotoxic T cell or natural killer T cell). The antibody or antibody fragment can interact with receptors on T cells (including, but not limited to, T cell receptors (TCRs)). The TCR can include TCRα, TCRβ, TCRγ, and / or TCRδ or TCRζ. The antibodies or antibody fragments of the present invention can bind to receptors on lymphocytes, dendritic cells, B cells, macrophages, monocytes, neutrophils, and / or NK cells. The antibodies or antibody fragments of the present invention can bind to cell surface receptors. The antibodies or antibody fragments of the present invention can bind to folate receptors. The antibodies or antibody fragments of the present invention can complex with T cell surface antigens (e.g., 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) or analogs or derivatives thereof, including, but not limited to). See, for example, U.S. Patent No. 6,479,470; WO2017 / 136659 and WO2014 / 153164 (each of which is incorporated herein by reference in its entirety).

[0147] In certain embodiments, the antibodies or antibody fragments described herein are anti-CD3 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-CD3 antibodies or antibody fragments or variants described herein can be humanized. Examples of the anti-CD3 antibodies or antibody fragments or variants described herein include, but are not limited to, CD3 analogs, isoforms, mimetics, fragments, or hybrids. The anti-CD3 antibodies, antibody fragments, or variants of the present invention include, but are not limited to, Fv, Fc, Fab and (Fab’)2, single-chain Fv (scFv), diabody, triabody, tetrabody, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules having other backbones, bispecific antibodies, and the like. The anti-CD3 antibodies or antibody fragments or variants of the present invention have the sequences of SEQ ID NOs: 1 to 62. The antibodies, fragments or variants of the present invention may be anti-CD3 Fab antibodies, fragments or variants. The antibodies, fragments or variants of the present invention may include one or more anti-CD3 Fabs. The antibodies, fragments or variants of the present invention may include two anti-CD3 Fabs. In certain embodiments, the anti-CD3 antibody has a heavy chain and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 1 to 62. In certain embodiments, the anti-CD3 antibody consists of a heavy chain and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 1 to 62. In certain embodiments, the anti-CD3 antibody has any one of the heavy chain amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57; and any one of the light chain amino acid sequences of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62.

[0148] Anti-CD3 bispecific antibodies containing non-natural amino acids are also described herein. In certain embodiments, anti-CD3 bispecific antibodies, antibody fragments, or variants include, but are not limited to, Fv, Fc, Fab and (Fab’)2, single-chain Fv (scFv), diabody, triabody, tetrabody, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules having other backbones, bispecific antibodies, and the like. In some embodiments, the anti-CD3 bispecific antibody or antibody fragment or variant is an anti-CD3 Fab bispecific antibody, fragment or variant containing one or more non-naturally encoded amino acids. The anti-CD3 Fab bispecific antibody or antibody fragment or variant of the present invention may have one or more of the sequences of SEQ ID NOs: 1-62. The bispecific antibody, fragment or variant of the present invention may be an anti-CD3 Fab antibody, fragment or variant. The anti-CD3 bispecific antibody may have a heavy chain and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 1-62. In some embodiments, the anti-CD3 antibody consists of a heavy chain and / or light chain amino acid sequence selected from the sequences of SEQ ID NOs: 1-62. In certain embodiments, the anti-CD3 bispecific antibody has any one of the heavy chain amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56, and 57; and any one of the light chain amino acid sequences of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. In some embodiments, the bispecific antibodies, fragments or variants described herein specifically bind to CD3. The bispecific antibodies, fragments or variants may be heteroreactive. The bispecific antibodies, fragments or variants may be heteroreactive with human and monkey antigens. In some embodiments, the antibody contains heteroreactive CDRs. The bispecific antibody may be a humanized antibody.

[0149]

Table 1

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[0151] JPEG2025098156000003.jpg64164

[0152] JPEG2025098156000004.jpg146164

[0153] JPEG2025098156000005.jpg208164

[0154] JPEG2025098156000006.jpg208164

[0155] JPEG2025098156000007.jpg218164

[0156] JPEG2025098156000008.jpg171164

[0157] 〔Non-natural amino acid〕 The present invention provides an anti-CD3 antibody, antibody fragment or variant containing one or more amino acids not naturally encoded. The introduction of one or more amino acids not naturally encoded into the anti-CD3 antibody can enable the application of complexation chemistry that does not react with 20 types of normal amino acids while involving specific chemical reactions with one or more amino acids not naturally encoded.

[0158] Some embodiments described herein are anti-CD3 antibodies that include one or more non-naturally encoded amino acids. The one or more non-natural amino acids can be encoded by a codon that does not encode one of the 20 natural amino acids. The one or more non-natural amino acids can be encoded by a nonsense codon (stop codon). The stop codon can be an amber codon. The amber codon can have a UAG sequence. The stop codon can be an ochre codon. The ochre codon can have a UAA sequence. The stop codon can be an opal codon or an amber (umber) codon. The opal codon or an amber (umber) codon can have a UGA sequence. The one or more non-natural amino acids can be encoded by a four-base codon.

[0159] Examples of one or more non-natural amino acids include, but are not limited to, p-azidophenylalanine (pAz), p-benzoylphenylalanine (pBpF), p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF), p-cyanophenylalanine (pCNF), p-carboxymethylphenylalanine (pCmF), 3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF), o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3-yl)alanine (HQA), (2,2'-bipyridin-5-yl)alanine (BipyA). The one or more non-natural amino acids can be β-amino acids (β3 and β2), homo-amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear nuclear amino acids, diamino acids, D-amino acids, N-methyl amino acids, or combinations thereof.In addition, examples of non-natural amino acids include, but are not limited to: (1) various substituted tyrosine and phenylalanine analogs (e.g., O-methyl-L-tyrosine, p-amino-L-phenylalanine, 3-nitro-L-tyrosine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine, p-isopropyl-L-phenylalanine, etc.); (2) amino acids having photo-crosslinkable aryl azide and benzophenone groups; (3) amino acids having unique chemical reactivity (e.g., acetyl-L-phenylalanine and m-acetyl-L-phenylalanine, O-allyl-L-tyrosine, O-(2-propynyl)-L-tyrosine, p-ethylthiocarbonyl-L-phenylalanine, p-(3-oxobutanoyl)-L-phenylalanine, etc.); (4) heavy atom-containing amino acids for phasing in X-ray crystallography (e.g., p-iodo and p-bromo-L-phenylalanine, etc.); (5) dihydroxy-L-phenylalanine which is a redox-active amino acid; (6) glycosylated amino acids (e.g., b-N-acetylglucosamine-O-serine and a-N-acetylgalactosamine-O-threonine, etc.); (7) fluorescent amino acids having naphthyl, dansyl, and 7-aminocoumarin side chains; (8) photocleavable and photoisomerizable amino acids having azobenzene and nitrobenzyl Cys, Ser, and Tyr side chains; (9) p-carboxymethyl-L-phenylalanine which is a phosphotyrosine mimic; (10) homoglutamine which is a glutamine homolog; and (11) 2-aminooctanoic acid. In some embodiments, the non-natural amino acid is N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In some embodiments, the non-natural amino acid has a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine.Examples of such amino acids also include those in which the natural N- or O-linkage between an amino acid and a sugar is replaced by a covalent bond not commonly found in nature. Such amino acids include, but are not limited to, alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include saccharides (2-deoxy-glucose, 2-deoxygalactose, etc.) not commonly found in natural proteins. Specific examples of non-natural amino acids include p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, and isopropyl-L-phenylalanine, etc., but are not limited to these. Further non-natural amino acids are disclosed in [Liu et al, Annu Rev Biochem, 79:413-44, 2010], [Wang et al, Angew Chem Int Ed, 44:34-66, 2005], and International Application Numbers PCT / US2012 / 039472, PCT / US2012 / 039468, PCT / US2007 / 088009, PCT / US2009 / 058668, PCT / US2007 / 089142, PCT / US2007 / 088011, PCT / US2007 / 001485, PCT / US2006 / 049397, PCT / US2006 / 047822 and PCT / US2006 / 044682 (each of which is hereby incorporated by reference in its entirety). In some embodiments, one or more non-natural amino acids may be p-acetylphenylalanine (pAF).

[0160] In certain embodiments of the present invention, an anti-CD3 antibody having one or more non-natural amino acids has one or more post-translational modifications. In one embodiment, the one or more post-translational modifications include the addition of a molecule. Molecules that can be added at this time include, but are not limited to, water-soluble polymers, polyethylene glycol derivatives, drugs, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, agents having biological activity, small molecules, or any combination of the above, or any other desired compound or substance. These molecules have a second reactive group and react with the one or more non-natural amino acids having a first reactive group. This reaction is carried out by chemical methods known to those skilled in the art as being suitable for specific reactive groups. For example, the first reactive group is an alkynyl moiety (for example, there is one in p-propargyloxyphenylalanine, a non-natural amino acid, and the propargyl group is sometimes also called an acetylene moiety, but is not limited thereto), and the second reactive group is an azide moiety. In this example, the chemical method of [3+2] cycloaddition is utilized. In other examples, the first reactive group is an azide moiety (for example, there is one in p-azido-L-phenylalanine of a non-natural amino acid, but is not limited thereto), and the second reactive group is an alkynyl moiety. In certain embodiments of the modified anti-CD3 antibody polypeptide of the present invention, in the sugar moiety having one or more post-translational modifications, one or more non-natural amino acids having one or more post-translational modifications (for example, non-natural amino acids having a keto functional group, but not limited thereto) are used. In certain embodiments, the post-translational modification is carried out in vivo in eukaryotic or non-eukaryotic cells. In other embodiments, the post-translational modification is carried out in vitro. In other embodiments, the post-translational modification is carried out both in vitro and in vivo.

[0161] In some embodiments, the non-natural amino acid may be modified to incorporate a chemical group. In some embodiments, the non-natural amino acid may be modified to incorporate a ketone group. One or more non-natural amino acids may include at least one oxime, carbonyl, dicarbonyl, hydroxylamine group, or combinations thereof. One or more non-natural amino acids may include at least one carbonyl, dicarbonyl, alkoxyamine, hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl / alkyl azide, norbornene, cyclopropene, trans-cyclooctene, or tetrazine functional group or combinations thereof.

[0162] In some embodiments described herein, non-natural amino acids are site-specifically incorporated into antibodies, antibody fragments or variants. In some embodiments, non-natural amino acids are site-specifically incorporated into anti-CD3 antibodies, antibody fragments or variants. Methods for incorporating non-natural amino acids into a molecule (e.g., a protein, polypeptide or peptide) are disclosed in U.S. Patent Nos. 7,332,571; 7,928,163; 7,696,312; 8,008,456; 8,048,988; 8,809,511; 8,859,802; 8,791,231; 8,476,411; or 9,637,411 (each incorporated herein by reference in its entirety), as well as in the examples herein. One or more non-natural amino acids can be incorporated by methods known in the art. For example, a cell-based or cell-free system can be used, or an auxotrophic strain can be used in place of engineered tRNA and synthetase. In certain embodiments, orthogonal tRNA synthetases are used as disclosed, for example, in PCT / US2002 / 012465; PCT / US2002 / 012635; PCT / US2003 / 032576; PCT / US2005 / 044041; PCT / US2005 / 043603; PCT / US2005 / 046618 (each incorporated herein by reference in its entirety). Incorporating one or more non-natural amino acids into an antibody or antibody fragment or variant can involve modifying one or more amino acid residues in the antibody or antibody fragment or variant. Modifying one or more amino acid residues in an antibody or antibody fragment or variant can involve mutating one or more nucleotides in the nucleotide sequence encoding the antibody or antibody fragment or variant. Mutating one or more nucleotides in the nucleotide sequence encoding an antibody or antibody fragment or variant can involve changing a codon encoding an amino acid to a nonsense codon. Incorporating one or more non-natural amino acids into an antibody or antibody fragment or variant can involve modifying one or more amino acid residues in the antibody or antibody fragment or variant to produce one or more amber codons in the antibody or antibody fragment or variant.One or more unnatural amino acids can be incorporated into an antibody or antibody fragment or variant in response to an amber codon. One or more unnatural amino acids can be site-specifically incorporated into an antibody or antibody fragment or variant. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can involve one or more genetically encoded unnatural amino acids having orthogonal chemical reactivity to the 20 standard amino acids for site-specifically modifying a bioactive molecule or targeting agent. Incorporating one or more unnatural amino acids can involve the use of a tRNA / aminoacyl-tRNA synthetase pair for site-specifically incorporating one or more unnatural amino acids at defined sites in a bioactive molecule or targeting agent in response to one or more amber nonsense codons. Additional methods for incorporating unnatural amino acids include, but are not limited to, the methods described in [Chatterjee et al, A Versatile Platform for Single- and Multiple-Unnatural Amino Acid Mutagenesis in Escherichia coli, Biochemistry, 2013], [Kazane et al, J Am Chem Soc, 135(1):340-6, 2013], [Kim et al, J Am Chem Soc, 134(24):9918-21, 2012], [Johnson et al, Nat Chem Biol, 7(11):779-86, 2011], and [Hutchins et al, J Mol Biol, 406(4):595-603, 2011]. One or more unnatural amino acids can be generated by the selective reaction of one or more natural amino acids. The selective reaction can be mediated by one or more enzymes. In a non-limiting example, one or more formylglycines can be generated by the selective reaction of one or more cysteines with formylglycine-generating enzyme (FGE) (as described in [Rabuka et al, Nature Protocols 7: 1052-1067, 2012]). One or more unnatural amino acids can be associated with a chemical reaction for forming a linker.The chemical reaction for forming the linker may include a bioorthogonal reaction. The chemical reaction for forming the linker may include click chemistry. See, for example, International Publication No. WO2006 / 050262, which is hereby incorporated by reference in its entirety.

[0163] [Molecule / drug having biological activity] Described herein are anti-CD3 antibodies or antibody fragments or variants thereof that include a molecule having biological activity linked to the antibody or fragment or variant via a non-natural amino acid. Molecules having biological activity include, but are not limited to, small molecules or agents, non-peptide compounds, drugs, second proteins or polypeptides or polypeptide analogs or derivatives, antibodies or antibody fragments or variants, agents having a second biological activity, targeting agents, or any combination of the foregoing or any other desirable compounds or substances. In some embodiments, the agent having biological activity is involved in the accumulation of cytotoxic T cells to cells (including, but not limited to, cancer cells or tumor cells). In some embodiments, the molecule having biological activity is a small molecule such as folic acid or its derivatives or analogs, or 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) or its derivatives or analogs (not limited thereto). In some embodiments, the molecule having biological activity is a folate or its derivatives or analogs. The molecule having biological activity can be selected from cell targeting molecules, ligands, proteins, peptides, peptoids, DNA aptamers, peptide nucleic acids, vitamins, substrates or substrate analogs, cholecystokinin B receptor, gonadotropin releasing hormone receptor, somatostatin receptor 2, avb3 integrin, gastrin releasing peptide receptor, neurokinin 1 receptor, melanocortin 1 receptor, neurotensin receptor, neuropeptide Y receptor, and C-type lectin-like molecule 1, receptors, co-receptors, transmembrane proteins, or cell markers or cell surface proteins. The molecule having biological activity can bind to a target cell. The molecule having biological activity can bind to a cell surface protein, cell surface marker, or cell surface molecule on a cell. The molecule having biological activity can bind to a cell surface molecule on a cell (such as, but not limited to, a cancer cell or tumor cell or immunosuppressive cell). The cell surface molecule can be a folate receptor molecule. The molecule having biological activity can be an agent that binds to prostate specific membrane antigen (PSMA) (such as, but not limited to, DUPA or its analogs or derivatives).A molecule or agent having biological activity can bind to cells that overexpress or highly express a cell surface marker, protein, or receptor.

[0164] In some embodiments, the molecule having biological activity can be a folate or folic acid ligand, or an analog or derivative thereof. The molecule having biological activity can bind to the folate receptor protein (FR). Such a molecule having biological activity can be N-(4-{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino}benzoyl)-L-glutamic acid (folic acid), or an analog or derivative thereof. The analog can be a folate-based moiety that preserves FR binding. The folic acid analog can preserve a substantial portion of the structure of folic acid. Further, the folic acid analog can be a slightly modified form of folic acid by virtue of being conjugated to a linker or an antibody or antibody fragment or variant. For example, the folic acid analog can be slightly modified by conjugating the folic acid carboxy group to a linker or an antibody or antibody fragment or variant. Further, folic acid can be slightly modified by virtue of being conjugated to a linker or an antibody or antibody fragment, while maintaining its FR binding properties. In some embodiments, the folic acid molecule targets folate receptor α. In some embodiments, the folic acid molecule targets folate receptor β. In some embodiments, folic acid or folate is used as a molecule or targeting agent having biological activity for binding to a folate receptor (FR) antigen that is overexpressed or highly expressed on an FR+ cell line. In some embodiments, the cells are cancer cells or immunosuppressive cells, but are not limited thereto.

[0165] A molecule or agent having biological activity can be site-specifically linked to one or more non-natural amino acids of an antibody or antibody fragment or variant by one or more linkers. The linker can be a chemical linker, a peptide linker, or a combination thereof. The chemical linker includes a linker via a non-natural amino acid. The chemical linker includes a linker via one or more non-natural amino acids. The chemical linker can include a chemical conjugate. The peptide linker has any amino acid sequence that links two amino acid sequences. The peptide linker can include 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. The peptide linker can be a part of any antibody, including domains of the antibody such as variable domains, CH1, CH2, CH3, and / or CL domains. The antibody or antibody fragment or variant can complex with a molecule having biological activity. The antibody or antibody fragment or variant can complex with a molecule having biological activity via a chemical linker and / or a peptide linker. In some embodiments, the present invention provides an anti-CD3 antibody or antibody fragment or variant complexed with one or more molecules or agents having biological activity. In some embodiments, the one or more molecules or agents having biological activity are one or more small molecules. In some embodiments, the present invention provides an anti-CD3 Fab antibody or antibody fragment or variant complexed with one or more small molecules. In some embodiments, the present invention provides an anti-CD3 Fab antibody or antibody fragment or variant complexed with one or more folic acid molecules. In some embodiments, the present invention provides an anti-CD3 Fab antibody or antibody fragment or variant complexed with one or more DUPA molecules. In some embodiments, the one or more folic acid molecules and / or the one or more DUPA molecules can complex with the anti-CDFab antibody via a chemical linker and / or a peptide linker.

[0166] 〔PEG linker / ligand / conjugate〕 The anti-CD3 antibody or antigen-binding polypeptide and the small molecule may be linked by a linker, a polymer, or a covalent bond. The linker, polymer, or small molecule may itself have a functional group that does not react with the 20 common amino acids. The linker or polymer may be a bifunctional linker or polymer. The bifunctional linker or polymer may be a branched linker or polymer. One or more bonds involved in the linkage of the anti-CD3 antibody or antigen-binding polypeptide to the bioactive molecule via a linker, polymer, or covalent bond may be irreversible, reversible, or labile under the desired conditions. One or more bonds involved in the linkage of the anti-CD3 antibody or antigen-binding polypeptide to the molecule via a linker, polymer, or covalent bond may regulate the release of the antigen-binding polypeptide or other molecule. The various small molecules may be made by chemical means, isolated as natural products, or made by other means by those skilled in the art.

[0167] Described herein are anti-CD3 antibodies or antibody fragments or variants comprising one or more non-naturally encoded amino acids linked to one or more water-soluble polymers (such as polyethylene glycol (PEG) molecules or ligands). Anti-CD3 antibodies or antibody fragments or variants comprising non-naturally encoded amino acids can be linked to two water-soluble polymers (such as two polyethylene glycol (PEG) molecules or ligands). Antibodies or antibody fragments or variants comprising non-naturally encoded amino acids and one or more bioactive molecules can be linked to one or more water-soluble polymers (such as polyethylene glycol (PEG) molecules or linkers). In some embodiments, antibodies or antibody fragments or variants comprising non-naturally encoded amino acids and two bioactive molecules are linked to two water-soluble polymers (such as polyethylene glycol (PEG) molecules or linkers).

[0168] This method may include linking an antibody or antibody fragment to a molecule having biological activity, or a water-soluble polymer, or a complex comprising a molecule having biological activity and a water-soluble polymer. This method may include complexing one or more linkers with a molecule having biological activity to generate a molecule-having-biological-activity-linker intermediate, and complexing the intermediate with an antibody or antibody fragment. This method may include complexing one or more linkers with a PEG molecule to generate a PEG-linker intermediate, and complexing the PEG-linker intermediate with an antibody or antibody fragment. This method may include complexing one or more linkers with an antibody or antibody fragment to generate an antibody-linker intermediate or an antibody-fragment-linker intermediate, and complexing the antibody-linker intermediate or the antibody-fragment-linker intermediate with another molecule having biological activity, a water-soluble polymer, or a complex comprising a molecule having biological activity and a water-soluble polymer. The methods described herein may include complexing one or more linkers with one or more antibodies or antibody fragments, one or more molecules having biological activity, or combinations thereof to generate one or more intermediates (such as antibody-linker intermediates, antibody-fragment-linker intermediates, and / or molecule-having-biological-activity-antibody complex-linker intermediates, etc.). This method may include complexing a first linker with an antibody or antibody fragment to generate an antibody-linker intermediate or an antibody-fragment-linker intermediate. This method may include complexing a linker with a molecule having biological activity to generate a molecule-having-biological-activity-linker intermediate.

[0169] The method for producing the bispecific anti-CD3 antibody complex of the present invention may include: (a) conjugating a first linker with an antibody or an antibody fragment containing one or more unnatural amino acids incorporated into the antibody or antibody fragment; (b) conjugating a second linker with a bioactive molecule to generate a bioactive molecule-linker intermediate; and (c) linking the two intermediates to generate an anti-CD3 antibody-bioactive molecule complex. The bioactive molecule can be a small molecule (e.g., a folic acid molecule or a DUPA molecule or analogs or derivatives thereof, but not limited thereto). In certain embodiments, the method for producing the bispecific anti-CD3 antibody complex of the present invention may include: (a) conjugating a first linker with an antibody or an antibody fragment containing one or more unnatural amino acids incorporated into the antibody or antibody fragment to generate an antibody-linker intermediate or an antibody fragment-linker intermediate; (b) conjugating a second linker with a bioactive molecule to generate a bioactive molecule-linker intermediate; and (c) linking the two intermediates to generate an anti-CD3 antibody-bioactive molecule complex. The bioactive molecule can be a small molecule (e.g., a folic acid molecule or a DUPA molecule or analogs or derivatives thereof, but not limited thereto). The method for producing the bispecific anti-CD3 Fab antibody-folic acid complex of the present invention may include: (a) conjugating a first linker with an antibody or an antibody fragment containing one or more unnatural amino acids incorporated into the anti-CD3 Fab antibody or antibody fragment to generate an antibody-linker intermediate or an antibody fragment-linker intermediate; (b) conjugating a second linker with a bioactive molecule to generate a bioactive molecule-linker intermediate; and (c) linking the two intermediates to generate an anti-CD3 Fab antibody-bioactive molecule complex. The bioactive molecule contains one or more folic acid molecules or DUPA molecules or analogs or derivatives. The linker contains one or more chemical linkers and / or peptide linkers. The linker contains one or more PEG molecules. The one or more PEG molecules are linear or branched PEG molecules. The one or more branched PEG molecules are bifunctional linkers.The average molecular weight of the PEG molecule is 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, or more. The average molecular weight of the PEG molecule is 5K, 10K or 20K.

[0170] Complexation of the antibody or antibody fragment, or a molecule having biological activity with one or more linkers can occur simultaneously. Complexation of the antibody or antibody fragment, or a molecule having biological activity with one or more linkers can occur sequentially. Complexation of the antibody or antibody fragment, or a molecule having biological activity with one or more linkers can occur in a single-step process (e.g., enzymatic complexation, or chemical process, or process or reaction, etc.). Complexation of the antibody or antibody fragment, or a molecule having biological activity with one or more linkers can occur in a two-step process (e.g., two enzymatic complexations, or two chemical processes, or two processes or two reactions, etc.). Complexation of the antibody or antibody fragment, or a molecule having biological activity with one or more linkers can occur in a process of two or more steps (e.g., two or more enzymatic complexations, or chemical processes, or processes or reactions, etc.).

[0171] Complexing the intermediate with an antibody or antibody fragment, or a molecule having biological activity, or a water-soluble polymer may involve oxime chemistry and / or click chemistry that form oxime bonds, as is well known to those skilled in the art. The antibody or antibody fragment may contain one or more unnatural amino acids. Linking the antibody or antibody fragment to the intermediate may involve forming an oxime between the unnatural amino acid and the linker intermediate. Complexing the linker with an antibody or antibody fragment, or a molecule having biological activity or a water-soluble molecule may involve ionic, covalent, non-covalent, or combinations thereof between the linker and the antibody or antibody fragment, or the molecule having biological activity or the water-soluble molecule. Complexation of an antibody or antibody fragment or a molecule having biological activity or a water-soluble molecule with a linker is known in the art. See, for example, [Roberts et al, Advanced Drug Delivery Reviews 54:459-476 (2002)].

[0172] Complexing one or more linkers with an antibody or antibody fragment and / or a molecule having biological activity may involve forming one or more oximes between the linker and the antibody or antibody fragment or the molecule having biological activity. Complexing one or more linkers with an antibody or antibody fragment and / or a molecule having biological activity may involve forming one or more stable bonds between the linker and the antibody or antibody fragment or the molecule having biological activity. Complexing one or more linkers with an antibody or antibody fragment and / or a molecule having biological activity may involve forming one or more covalent bonds between the linker and the antibody or antibody fragment or the molecule having biological activity. Complexing one or more linkers with an antibody or antibody fragment and / or a molecule having biological activity may involve forming one or more non-covalent bonds between the linker and the antibody, antibody fragment or the molecule having biological activity. Complexing one or more linkers with an antibody or antibody fragment and / or a ligand may involve forming one or more ionic bonds between the linker and the antibody or antibody fragment or the molecule having biological activity.

[0173] The complexing of one or more linkers with an antibody or antibody fragment can include site-specifically complexing one or more linkers with the antibody or antibody fragment. Site-specific conjugation can include linking one or more linkers to non-natural amino acids of the antibody or antibody fragment. Linking one or more linkers to non-natural amino acids of the antibody or antibody fragment can include oxime formation. Linking one or more linkers to non-natural amino acids of the antibody or antibody fragment can include sulfide formation. Linking one or more linkers to non-natural amino acids of the antibody or antibody fragment can include, by way of non-limiting example, reacting the hydroxylamine of one or more linkers with the aldehyde or ketone of an amino acid. The amino acid may be a non-natural amino acid. Linking one or more linkers to non-natural amino acids of the antibody or antibody fragment can include, by way of non-limiting example, reacting the bromo derivative of one or more linkers with the thiol of an amino acid. The amino acid may be a non-natural amino acid.

[0174] One or more PEGs or linkers can include disulfide bridges that link two cysteine residues using conjugation chemistry, as known to those of skill in the art. (See, e.g., [ThioBridge™ technology, Abzena] as well). Two or more PEG molecules or linkers can include maleimide bridges that link two amino acid residues. The two amino acids may be located at the C-terminus of the antibody or antibody fragment. One or more linkers can include maleimide bridges that link two cysteine residues. The two cysteine residues may be at the C-terminus of the antibody or antibody fragment. In some embodiments, one or more PEGs can be C-terminal PEG conjugates. In some embodiments, the C-terminal PEG molecule may not involve a covalent disulfide bond or addition between the heavy chain antibody and the light chain antibody, or between antibody fragments. In some embodiments, two or more PEG molecules located at the C-terminus can be covalently bonded or crosslinked between the heavy and light chains of the antibody or antibody fragment. Such PEG conjugates or linkers are described herein.

[0175] In some embodiments described herein, the folic acid-PEG linker comprises one or more folic acid molecules and one or more PEG molecules. The one or more PEG molecules can be 5 kDa, 10 kDa, 15 kDa, 20 kDa, or more. The PEG molecules include both linear polymers and branched polymers, and the average molecular weight is from 0.1 kDa to 100 kDa. In some embodiments, the molecular weight of the poly(ethylene glycol) molecule or linker is from about 0.1 kDa to about 100 kDa. In some embodiments, the molecular weight of the poly(ethylene glycol) molecule or linker is from 0.1 kDa to 50 kDa. In some embodiments, the poly(ethylene glycol) molecule or linker is a branched polymer or a branched linker. In some embodiments, the molecular weight of each branch of the poly(ethylene glycol) branched polymer or linker is from 1 kDa to 100 kDa, or from 1 kDa to 50 kDa. PEG molecules are well known in the art, for example, see the catalog ‘‘Polyethylene Glycol and Derivatives for Biomedical Applications’’ (2001) of Shearwater Corporation.

[0176] In certain embodiments described herein, the anti-CD3 Fab-folic acid PEGylated conjugate comprises one or more PEG molecules. In certain embodiments described herein, the anti-CD3 Fab-folic acid PEGylated conjugate comprises one or more C-terminal PEG molecules. In some embodiments, the C-terminal PEG molecule may not involve a covalent disulfide bond or addition between the heavy chain antibody or antibody fragment and the light chain antibody or antibody fragment. In some embodiments, the C-terminal PEG molecule can be added separately to the heavy and light chains of the antibody or antibody fragment. In some embodiments, two or more C-terminal PEG molecules can be covalently bonded or crosslinked between the heavy and light chains of the antibody or antibody fragment. In some embodiments, two or more PEG molecules can be covalently bonded or crosslinked via a maleimide bridge that links two cysteine residues.

[0177] PEGylation can be used to improve pharmacokinetics and modulate the cytotoxicity of compositions. Since the PEG moiety adds a significant hydrodynamic radius to the protein, PEGylation of the protein can increase its serum half-life by retarding renal clearance. By the method of covalently attaching the hydrophilic polymer poly(ethylene glycol) (abbreviated PEG), (i) water solubility and bioavailability are improved, (ii) serum half-life and therapeutic half-life are increased, (iii) immunogenicity and biological activity are modulated, or (iv) for molecules with multiple biological activities (such as proteins, peptides, and particularly hydrophobic molecules), the circulation time is extended. PEG has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, removal of toxicity, and removal of immunogenicity are important. Preferably, PEGylation does not change or only minimally changes the activity of the bioactive molecule. Preferably, the increase in half-life is greater than any decrease in biological activity. [Rader et al. in Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5396-400] discloses a method of conferring effector function to a synthetic small molecule and extending its serum half-life by reacting it with a generic antibody molecule (incorporated herein by reference). The conjugate disclosed in the above literature was created by reversibly covalently linking between mAb 38C2 (a catalytic antibody mimicking the natural aldolase enzyme) and a diketone derivative of an Arg-Gly-Asp peptide mimetic targeting integrin via a reactive lysine residue on the antibody. Not only was the half-life of the peptide mimetic extended, but a switch in antibody targeting was observed such that the conjugate selectively targeted the cell surface expressing integrin α v β3 and α v β5.

[0178] In some embodiments, an anti-CD3 antibody having a non-naturally encoded amino acid is linked to a water-soluble polymer (such as polyethylene glycol (PEG)) via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody having a non-naturally encoded amino acid is linked to folate via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody having a non-naturally encoded amino acid is linked to a folate derivative via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody having a non-naturally encoded amino acid is linked to a water-soluble polymer (such as polyethylene glycol (PEG)) via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody having a non-naturally encoded amino acid is linked to a water-soluble polymer derivative (such as a polyethylene glycol (PEG) derivative) via the side chain of the non-naturally encoded amino acid. In some embodiments, a water-soluble polymer-folate linker is provided, and an anti-CD3 antibody having one or more non-naturally encoded amino acids is linked to folate and / or a water-soluble polymer and / or a linker via the side chain of the one or more non-naturally encoded amino acids.

[0179] In some embodiments, the folate moiety is derived from the following structures, including the structures shown in FIGS. 12A-12F:

[0180]

Chemical formula

[0181] In some embodiments, the water-soluble polymer is poly(ethylene glycol). In some embodiments, the term poly(ethylene glycol) includes any form of poly(ethylene glycol) including linear poly(ethylene glycol), branched poly(ethylene glycol), bifunctional poly(ethylene glycol), multi-arm poly(ethylene glycol), derivatized poly(ethylene glycol), and branched poly(ethylene glycol).

[0182] The molecular weight of the poly(ethylene glycol) may be from 1 kDa to 100 kDa. The molecular weight of the poly(ethylene glycol) can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 kDa. The molecular weight of the poly(ethylene glycol) can be from about 1 kDa to about 25 kDa, or from about 5 kDa to 20 kDa. The molecular weight of the poly(ethylene glycol) can be about 5 kDa, or about 10 kDa, or about 20 kDa. The molecular weight of the poly(ethylene glycol) may be 5 kDa or 10 kDa or 20 kDa. The molecular weight of the poly(ethylene glycol) may be 5 kDa.

[0183] In some embodiments, the bifunctional water-soluble polymer-folic acid linker has the following structure:

[0184]

Chemical formula

[0185] wherein, A has the following structure:

[0186]

Chemical formula

[0187] B is a divalent group that links A and C; C and E are each independently -alkylene-, -alkylene-C(O)-, -(alkylene-O)n’ -alkylene-, -(alkylene-O) n’ -alkylene-C(O)-, -(alkylene-O) n’ -(CH2) n’ -NHC(O)-(CH2) n’ -C(Me)2-S-S-(CH2) n’ -NHC(O)-(alkylene-O) n’ -alkylene-, -(alkylene-O) n’ -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n’ selected from the group consisting of -alkylene-U-alkylene-, wherein n' is independently an integer of 1 or more; D is a trivalent group that connects C, F, and E; F is a water-soluble polymer such as polyethylene glycol (PEG); wherein, Y is selected from the group consisting of hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, ketoamine, ketoalkyne, alkyne, cycloalkyne, and enedione.

[0188]

[0189] In some embodiments, B is a substituted divalent heterohydrocarbyl residue. In some embodiments, the substituent includes one or more carboxyl, ketone, and / or amide functional groups. In some embodiments, the heteroatom is selected from N, O, and S.

[0190]

Chemical formula

[0191] ​In some embodiments, D is a substituted trivalent heterohydrocarbyl residue. In some embodiments, the substituent includes one or more carboxyl, ketone, and / or amide functional groups. In some embodiments, the heteroatom is selected from N, O, and S.

[0192] In some embodiments, D has the following structure:

[0193]

Chemical formula

[0194] In some embodiments, C and E are each -(alkylene - O) n’ - alkylene -. In some embodiments, each alkylene is -CH2CH2-.

[0195] In some embodiments, n' is 1 to 20, or 1 to 10, or 1 to 5.

[0196] In some embodiments, F has the following structure:

[0197]

Chemical formula

[0198] wherein n is 2 to 10,000. In some embodiments, n is selected such that the molecular weight of poly(ethylene glycol) (PEG) is 1 kDa to 100 kDa. For example, the molecular weight can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 kDa. For example, the molecular weight can be about 1 kDa to about 25 kDa, or about 5 kDa to 20 kDa. For example, the molecular weight can be about 5 kDa, or about 10 kDa, or about 20 kDa. For example, the molecular weight can be 5 kDa, or 10 kDa, or 20 kDa. For example, the molecular weight can be 5 kDa.

[0199] In some embodiments, the bifunctional water-soluble polymer-folic acid linker has the following structure:

[0200] [Chemical formula]

[0201] In some embodiments, an anti-CD3 antibody comprising at least one non-naturally encoded amino acid is linked to a water-soluble polymer bifunctional PEG-folic acid linker and thus has the following structure:

[0202] [Chemical formula]

[0203] Wherein A, B, C, D, E and F are as defined in any of the above embodiments, and Z is an oxime or cyclic bond linked to the anti-CD3 antibody via a non-natural amino acid.

[0204] In some embodiments, Z has the following structure:

[0205] [Chemical formula]

[0206] Wherein J is of arbitrary configuration and, if present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; G is of arbitrary constitution and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -(where k is 1, 2 or 3), -S(O) k (alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O) k N(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O) k N(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-(where each R’ is independently H, alkyl, or substituted alkyl) and is a linker selected from the group consisting of; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; wherein R1 and / or R2 is an anti-CD3 antibody; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or, optionally, R3 and R4 or two R3 groups form cycloalkyl or heterocycloalkyl.

[0207] In some embodiments, Z has the following structure:

[0208]

Chemical formula

[0209] wherein J, G, R1, R2, R3, and R4 are as defined above, and where D has the following structure:

[0210]

Chemical formula

[0211] wherein each R 17 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaliaryl, substituted alkaliaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R'')2, -(alkylene or substituted alkylene)-C(O)SR'', -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R'', -C(O)2R'', or -C(O)N(R'')2 (where each R'' is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaliaryl, substituted alkaliaryl, aralkyl, or substituted aralkyl); each Z1 is a bond, CR 17 R 17 , O, S, NR', CR 17 R 17 -CR 17 R 17 , CR 17 R 17 -O, O-CR 17 R17 、CR 17 R 17 -S, S-CR 17 R 17 、CR 17 R 17 -NR’, or NR’-CR 17 R 17 ; and each R’ is H, alkyl, or substituted alkyl; each Z2 is selected from the group consisting of a bond, -C(O)-, -C(S)-, optionally substituted C1-C3 alkylene, optionally substituted C1-C3 alkenylene, and optionally substituted heteroalkyl; each Z3 is independently selected from the group consisting of a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, -O-, -S-, -C(O)-, -C(S)-, and -N(R’)-; each T3 is a bond, C(R’’)(R’’), O, or S (provided that when T3 is O or S, R’’ is not halogen); each R’’ is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; m and p are 0, 1, 2, or 3 (provided that at least one of m or p is not 0); M2 has the following structure,

[0212]

Chemical formula

[0213] wherein (a) represents a bond to the B group and (b) represents a bond to each position within the heterocyclic group; M3 has the following structure,

[0214]

Chemical formula

[0215] In the formula, (a) represents the bond to the B group, and (b) represents the bonds to the respective positions within the heterocyclic group; M4 has the following structure,

[0216]

Chemical formula

[0217] In the formula, (a) represents the bond to the B group, and (b) represents the bonds to the respective positions within the heterocyclic group; Each R 19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, halogenated alkyl, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; Each R 16 is independently selected from the group consisting of hydrogen, halogen, alkyl, NO2, CN and substituted alkyl.

[0218] The present invention provides a highly efficient method for selectively modifying proteins with PEG derivatives. This method is related to a method for selectively incorporating non-genetically encoded amino acids. Non-genetically encoded amino acids include amino acids having functional groups or substituents (such as, but not limited to, a ketone moiety, an azide moiety or an acetylene moiety) that are not found in the 20 amino acids normally incorporated into proteins corresponding to the selected codons (however, not limited thereto). As a result, the amino acids react appropriately with PEG derivatives and are modified. Once incorporated, the amino acid side chains can then be modified with respect to certain functional groups or substituents present in the naturally encoded amino acids by appropriate chemical methods known to those skilled in the art. Various known chemical methods are preferably used in the present invention for incorporating water-soluble polymers into proteins. Such methods include, but are not limited to, the Huisgen [3+2] cycloaddition reaction (see, for example, [Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109, 1991; Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, Ed. Padwa, A., Wiley, New York, p. 1-176, 1984]) between an acetylene derivative or an azide derivative (however, not limited thereto), respectively.

[0219] The Huisgen [3+2] cycloaddition is related to cycloaddition rather than nucleophilic substitution reaction, so proteins can be modified with very high selectivity. This reaction can be carried out under aqueous conditions at room temperature with excellent site specificity (1,4>1,5) by adding a catalytic amount of Cu(I) salt to the reaction mixture (for example, [Tornoe, et al., Org. Chem. 67:3057-3064, 2002; Rostovtsev, et al., Angew. Chem. Int. Ed. 41:2596-2599, 2002] and WO 03 / 101972). Molecules that can be added to the proteins of the present invention by [3+2] cycloaddition include substantially any molecule having a suitable functional group or substituent (including, but not limited to, azide derivatives or acetylene derivatives). These molecules can be added to non-natural amino acids having an acetylene group (for example, but not limited to, p-propargyloxyphenylalanine) or non-natural amino acids having an azide group (for example, but not limited to, p-azido-phenylalanine), respectively.

[0220] The resulting five-membered ring from the Huisgen [3+2] cycloaddition is generally irreversible in a reducing environment and is also stable against hydrolysis for a long time in an aqueous environment. Therefore, even in a harsh aqueous environment, the physical and chemical properties of a wide variety of substances can be modified by the activated PEG or PEG derivatives of the present invention. More importantly, since the azide moiety and the acetylene moiety are specific to each other (and, for example, do not react with any of the 20 common amino acids encoded by genes), proteins can be modified with very high selectivity at one or more specific sites.

[0221] The present invention also provides water-soluble and hydrolysis-stable derivatives of PEG derivatives and related hydrophilic polymers having one or more acetylene moieties or azide moieties. PEG polymer derivatives having an acetylene moiety couple with an azide moiety selectively introduced into a protein corresponding to a selected codon with high selectivity. Similarly, PEG polymer derivatives having an azide moiety couple with an acetylene moiety selectively introduced into a protein corresponding to a selected codon with high selectivity.

[0222] More specifically, examples of the azide moiety include, but are not limited to, alkyl azides, aryl azides, and derivatives of these azides. Derivatives of alkyl azides and aryl azides may have other substituents as long as the reactivity specific to acetylene is preserved. Examples of the acetylene moiety include alkyl acetylenes, aryl acetylenes, and derivatives of these. Derivatives of alkyl acetylenes and aryl acetylenes may have other substituents as long as the reactivity specific to azide is preserved.

[0223] Thus, it is intended that the anti-CD3 antibody encompasses any polypeptide that exhibits specific binding ability to a target molecule or antigen. Any known antibody or antibody fragment is an anti-CD3 antibody.

[0224] In one embodiment, a composition of an anti-CD3 antibody having a non-natural amino acid (such as p-(propargyloxy)-phenylalanine) is provided. Also provided are various compositions comprising p-(propargyloxy)-phenylalanine and further comprising a protein and / or a cell (but not limited thereto). In one aspect, a composition containing p-(propargyloxy)-phenylalanine, which is a non-natural amino acid, further comprises an orthogonal tRNA. The non-natural amino acid may be bound to the orthogonal tRNA (for example, but not limited to, by a covalent bond). Examples of such bonds include (i) a covalent bond with an orthogonal tRNA via an amino-acyl bond, (ii) a covalent bond with the 3'OH or 2'OH of the ribose sugar at the end of the orthogonal tRNA, etc. (but not limited thereto).

[0225] [Measurement of the activity and affinity of an anti-CD3 antibody for the antigen or binding target of the anti-CD3 antibody] The activity of the anti-CD3 antibody can be measured by standard in vitro or in vivo assays. For example, a cell or cell line that binds to the anti-CD3 antibody (for example, a cell containing a natural anti-CD3 antibody antigen or binding target, or a cell that recombinantly produces an anti-CD3 antibody antigen or binding target, but not limited thereto) can be used to evaluate the binding of the anti-CD3 antibody. For an unPEGylated antigen-binding polypeptide or a PEGylated antigen-binding polypeptide having a non-natural amino acid, the affinity of the anti-CD3 antibody for the antigen or binding target of the anti-CD3 antibody can be measured using known techniques (for example, BIAcore (trademark) biosensor (Pharmacia) or Octet (ForteBio), etc.).

[0226] Regardless of the method employed to produce the anti-CD3 antibody, the anti-CD3 antibody is subjected to an assay for evaluating biological activity. When appropriate, a tritiated thymidine assay may be performed to confirm the degree of cell division. However, other biological assays may also be used to confirm the desired activity. For example, the activity of the anti-CD3 antibody is also shown by a biological assay that measures the ability to inhibit the biological activity (such as enzyme activity, proliferative activity, or metabolic activity) of the antigen. Other in vitro assays known to those skilled in the art may also be used to confirm biological activity. Generally, when appropriate for the biological activity of the antigen, the biological activity test must provide an analysis of the desired result. This analysis may be, for example, an increase or decrease in biological activity (compared to the unmodified anti-CD3 antibody), a change in biological activity (compared to the unmodified anti-CD3 antibody), an affinity analysis with the receptor, a conformational or structural change, or an analysis of the serum half-life. Those skilled in the art will also be aware of other assays useful for testing the desired final result.

[0227] [Measurement of Efficacy, Functional Half-Life in Vivo, and Pharmacokinetic Parameters] An important aspect of the present invention is the extension of the biological half-life obtained by the construction of anti-CD3 antibodies (including those with and without the complexation of the anti-CD3 antibody with a water-soluble polymer moiety). Since the serum concentration of the anti-CD3 antibody rapidly decreases, the evaluation of the biological response to treatment with complexed or non-complexed anti-CD3 antibodies and their variants has been important. Preferably, the complexed or non-complexed anti-CD3 antibodies and their variants of the present invention extend the serum half-life even after intravenous administration. For this reason, measurement by, for example, ELISA or a primary screening assay becomes possible. The measurement of the biological half-life in vivo is performed by the methods described herein.

[0228] The pharmacokinetic parameters of an antigen-binding polypeptide having non-naturally encoded amino acids can be evaluated by normal Sprague-Dawley rats (male). The pharmacokinetic data of anti-CD3 antibodies have been well studied in several species and can be directly compared with the data obtained for anti-CD3 antibodies having non-naturally encoded amino acids.

[0229] The specific activities of the anti-CD3 antibodies according to the present invention can be measured by various known assays. The biological activities of the anti-CD3 antibody mutant proteins or fragments thereof according to the present invention, whether obtained or purified, can be tested by the methods described or incorporated herein or methods known to those skilled in the art.

[0230] [Administration and Pharmaceutical Compositions] The polypeptides or proteins of the present invention (such as, but not limited to, anti-CD3 antibodies, synthetic enzymes, proteins having one or more non-natural amino acids) may optionally be employed for therapeutic use (for example, in combination with a suitable pharmaceutical carrier, but not limited thereto). Such compositions include, for example, a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, physiological saline, buffered saline, dextrose, water, glycerol, ethanol, and / or combinations thereof. The formulation is made to suit the mode of administration. Generally, methods of administering proteins are well known in the art and can also be applied to the administration of the polypeptides of the present invention.

[0231] A therapeutic composition comprising one or more of the polypeptides of the present invention may optionally be tested in one or more in vitro and / or in vivo animal models of a suitable disease to confirm efficacy and metabolism in tissues, or to evaluate the dosage. This test follows methods well-known in the art. Specifically, the dosage can be initially determined by activity, stability, or other appropriate measures (i.e., comparative assays) against natural amino acid homologs of the unnatural amino acids herein (e.g., but not limited to, a comparison of an anti-CD3 antibody modified to have one or more unnatural amino acids with an anti-CD3 antibody consisting of natural amino acids).

[0232] Administration can be by any route normally used for introduction such that the molecule ultimately contacts the blood or tissue cells. The unnatural amino acid polypeptide of the present invention is administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers in any configuration. Suitable methods for administering the polypeptide according to the present invention to a patient are available. Also, a particular route is usually more immediate and can result in a more effective action or response than other routes (however, two or more routes can be used for administration of a particular composition).

[0233] The pharmaceutically acceptable carrier is determined to some extent by the particular composition to be administered and the particular method used for administration of that composition. Therefore, there are various types of suitable formulations of the pharmaceutical composition of the present invention.

[0234] The polypeptide composition can be administered by various routes. Such routes include, but are not limited to, oral administration, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, topical administration, sublingual administration, or rectal administration. A composition comprising an (modified or unmodified) unnatural amino acid polypeptide can also be administered using liposomes. Such administration routes and suitable formulations are well-known to those skilled in the art.

[0235] An anti-CD3 antibody having a non-natural amino acid can also be formulated as an aerosol preparation for administration by inhalation, either alone or in combination with other suitable components (i.e., it can be "sprayed"). The aerosol preparation can be placed in a pressurizable propellant (e.g., dichlorodifluoromethane, propane, nitrogen, etc.).

[0236] Formulations suitable for parenteral administration (e.g., intra-articular route (into the joint), intravenous route, intramuscular route, intradermal route, intraperitoneal route, and subcutaneous route, etc.) include aqueous or non-aqueous, isotonic sterile injection solutions. This injection solution may include antioxidants, buffers, bacteriostatic agents, and solutes that maintain the formulation isotonic with the blood of the subject patient, as well as aqueous and non-aqueous sterile suspensions (which may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, preservatives). The packaged anti-CD3 antibody formulation may be enclosed in a container (such as ampoules and vials), with each dose being enclosed separately or multiple doses being enclosed.

[0237] Parenteral administration and intravenous administration are preferred routes of administration. In particular, the routes of administration already used in the treatment with natural amino acid homologs (e.g., the routes typically used in EPO, GH, anti-CD3 antibodies, G-CSF, GM-CSF, IFN, interleukins, antibodies, and / or any other pharmaceutically delivered proteins, but not limited to these) provide suitable routes of administration and dosage forms for the polypeptides of the present invention in combination with the dosage forms used with those routes of administration.

[0238] Regarding the present invention, the dosage to be administered to a patient is an amount sufficient to cause a beneficial therapeutic response in the patient over a long period of time. Alternatively, depending on the use, it may also be an amount to prevent infection by a pathogen or an amount to inhibit other appropriate activities (however, not limited thereto). The dosage is determined according to (i) the effect of a specific vector or formulation, (ii) the activity, stability or serum half-life of the non-natural amino acid polypeptide employed, (iii) the condition of the patient, and (iv) the weight or body surface area of the patient to be treated. Also, the single dose is determined according to the life and constitution of a specific patient and the degree of any unfavorable side effects associated with the administration of a specific vector, formulation, etc.

[0239] In determining the effective amount of a vector or formulation to be administered in the treatment or prevention of a disease (such as, but not limited to, cancer, genetic diseases, diabetes, AIDS, etc.), the level in circulating plasma, the toxicity of the formulation, the progression of the disease, and / or, if relevant, the production of anti-non-natural amino acid polypeptide antibodies are evaluated by a physician.

[0240] For example, the dosage to be administered to a 70 kg patient is usually within a range equivalent to the dosage of currently used therapeutic proteins and is adjusted according to the changed activity or serum half-life of the present composition. The vectors of the present invention can supplement the treatment of the condition by any known therapy. Such known therapies include the administration of antibodies, the administration of vaccines, and the administration of cytotoxic substances, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, etc.

[0241] In administering the formulation of the present invention, it is administered at a pace determined by the LD-50 or ED-50 of the formulation and / or at a pace determined by observing any side effects caused by non-natural amino acids at various concentrations. In the latter case, the observation results may be applied to the weight and overall health condition of the patient, but not limited thereto. The administration may be completed with a single dose, or the dosage may be divided and the administration completed.

[0242] If a patient receiving the preparation complains of fever, chills or myalgia, administer an appropriate amount of aspirin, ibuprofen, acetaminophen or other antipyretic analgesics. For patients showing reactions such as fever, myalgia and chills to the injection, pre-medicate with any one of, but not limited to, aspirin, acetaminophen or diphenhydramine, etc. 30 minutes before the next injection. For more severe chills and myalgia where no prompt response to antipyretic agents and antihistamines is seen, use meperidine. Depending on the severity of the reaction, slow down or stop the pace of cell injection.

[0243] The human antigen-binding polypeptide of the present invention can be administered directly to a mammalian subject. Administration can be by any route commonly used for the introduction of anti-CD3 antibodies into a subject. The anti-CD3 antibody composition according to an embodiment of the present invention includes those suitable for oral administration, rectal administration, topical administration, inhalation administration (such as, but not limited to, by aerosol), buccal administration (including, but not limited to, sublingual administration), intravaginal administration, parenteral administration (such as subcutaneous administration, intramuscular administration, intradermal administration, intra-articular administration, intra-thoracic administration, intraperitoneal administration, intracerebral administration, intra-arterial administration or intravenous administration, etc., but not limited to these), topical administration (i.e., administration to the skin surface and mucosal surface, including administration to the airway surface), and transdermal administration. However, in any given case, the optimal route depends on the nature and severity of the condition to be treated. Administration can be either local or systemic. The preparation of the compound may be stored in a sealed container (such as ampoules and vials), either in a single-dose amount or in multiple-dose amounts. The anti-CD3 antibody of the present invention can be prepared as a mixture in an injectable form in a single-dose amount, together with a pharmaceutically acceptable carrier (such as, but not limited to, a solution, suspension or emulsion). Further, the anti-CD3 antibody of the present invention may be administered by continuous infusion (such as, but not limited to, using a small pump such as an osmotic pump), as a single bolus dose, or as a sustained-release depot.

[0244] Formulations suitable for administration include aqueous or non-aqueous solutions (sterile isotonic solutions), which may contain antioxidants, buffers, bacteriostatic agents, and solutes to maintain the isotonicity of the formulation. Aqueous or non-aqueous sterile suspensions are also suitable formulations for administration, which may contain suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives. Solutions and suspensions can be prepared from sterile powders, sterile granules, and tablets of the above-mentioned types.

[0245] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is determined to some extent by the particular composition to be administered and the particular method of administration of the composition. Therefore, there are various types of suitable formulations of the pharmaceutical composition of the present invention (optionally including a pharmaceutically acceptable carrier, excipient, or stabilizer) (see, for example, [Remington's Pharmaceutical Sciences, 17 th ed. 1985]).

[0246] Suitable carriers include the following: buffers (phosphates, borates, HEPES, citrates, and other organic acids); antioxidants (such as ascorbic acid); low molecular weight polypeptides (less than about 10 residues); proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); monosaccharides, disaccharides, and other hydrocarbons (such as glucose, mannose, or dextrin); chelating agents (such as EDTA); divalent metal ions (such as zinc, cobalt, or copper); sugar alcohols (such as mannitol or sorbitol); counterions forming salts (such as sodium); and / or nonionic surfactants (such as Tween™, Pluronics™, or PEG).

[0247] The anti-CD3 antibodies of the present invention (including antibodies linked to water-soluble polymers (such as PEG)) can be administered as a sustained release system (or as a partially sustained release system). Examples of the sustained release composition include, but are not limited to, molded articles of semipermeable polymer bases (such as films or microcapsules). Examples of the sustained release base include the following biocompatible substances: poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 1981; Langer, Chem. Tech., 12: 98-105 1982); ethylene-vinyl acetate (Langer et al., supra); or, poly-D-(-)-3-hydroxybutyric acid (EP133,988); polylactide (polylactic acid) (U.S. Patent No. 3,773,919, EP58,481); polyglycolide (a polymer of glycolic acid); polylactide-co-glycolide anhydride polymer (a copolymer of lactic acid and glycolic acid); copolymer of L-glutamic acid and γ-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 22, 547-556 1983); poly(ortho)esters; polypeptides; hyaluronic acid; collagen; chondroitin sulfate; carboxylic acids; fatty acids; phospholipids; polysaccharides; nucleic acids; polyamino acids; amino acids (such as phenylalanine, tyrosine, isoleucine, etc.); polynucleotides; polyvinylpropylene; polyvinylpyrrolidone; and silicones. Further, the sustained-release composition may further contain a compound encapsulated in liposomes. Liposomes containing the compound are prepared by methods known per se. See DE3,218,121, [Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692, 1985], [Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034, 1980], EP52,322, EP36,676, EP88,046, EP143,949, EP142,641, Japanese Patent Application Sho 83-118008, U.S. Patent No. 4,485,045, No. 4,544,545, EP102,324 (all publications and patent documents incorporated by reference are hereby incorporated herein by reference).

[0248] The anti-CD3 antibody encapsulated in liposomes can be prepared, for example, by the methods described in the following documents: DE3,218,121, [Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692, 1985], [Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034, 1980], EP52,322, EP36,676, EP88,046, EP143,949, EP142,641, Japanese Patent Application No. 83-118008, U.S. Patent Nos. 4,485,045, 4,544,545, EP102,324. The composition and size of liposomes are well known and can also be easily determined empirically by those skilled in the art. Some examples regarding liposomes are described, for example, as follows: [Park JW, et al., Proc. Natl. Acad. Sci. USA 92:1327-1331, 1995], [Lasic D and Papahadjopoulos D (eds): Medical Applications of Liposomes, 1998], [Drummond DC, et al., Liposomal drug delivery systems for cancer therapy, in Teicher B (ed): Cancer Drug Discovery and Development, 2002], [Park JW, et al., Clin. Cancer Res. 8:1172-1181, 2002], [Nielsen UB, et al., Biochim. Biophys. Acta 1591(1-3):109-118, 2002, Mamot C, et al., Cancer Res. 63: 3154-3161, 2003] (all publications and patent documents cited are hereby incorporated by reference into this specification).

[0249] Regarding the present invention, the dosage administered to a patient must be an amount sufficient to cause an advantageous response in the subject over a long period of time. When the anti-CD3 antibody of the present invention is administered parenterally, the total pharmaceutically effective amount per dosage is usually about 0.01 μg / kg / day to about 100 μg / kg, or about 0.05 mg / kg to about 1 mg / kg (per patient body weight). However, the dosage is left to the therapeutic judgment. Also, the dosing frequency is left to the therapeutic judgment. It may be administered more frequently or less frequently than commercially available anti-CD3 antibody products approved for use in humans. The bispecific antigen-binding polypeptide of the present invention can usually be administered by any of the above-described administration routes.

[0250] [Therapeutic Use of the Anti-CD3 Antibody Bioconjugate of the Present Invention] The anti-CD3 antibody polypeptide of the present invention is useful for treating a wide range of disorders. The anti-CD3 antibody composition described herein can be used to modulate the immune response. Modulation of the immune response can include stimulating, activating, enhancing, or upregulating the immune response. Modulation of the immune response can include suppressing, inhibiting, preventing, reducing, or downregulating the immune response. The described anti-CD3 antibody-folic acid composition can have advantages in that the natural folic acid ligand penetrates solid tumors while targeting, for example, folate receptor α on tumors and folate receptor β on immunosuppressive cells. In some embodiments, the tumor is a liquid or solid tumor.

[0251] Disclosed herein is a method of treating a condition of a subject using the anti-CD3 antibody complex or pharmaceutical composition of the present invention. In some cancers, overexpression of certain cell surface receptors allows for selective targeting of cancer cells with small molecules or drugs while minimizing effects on healthy cells. For example, 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA), which targets prostate-specific membrane antigen (PMSA), can be conjugated to a surface antigen (anti-CD3) binding antibody of T cells to selectively mobilize or target cytotoxic T cells to kill the prostate. N-(4-{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl}benzoyl)-L-glutamic acid (folic acid) can also be used as a biologically active molecule that binds to the folic acid receptor (FR) antigen overexpressed on FR+ cancer cell lines.

[0252] The present invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. The cancer can be ovarian cancer (including, but not limited to, epithelial tumors, stromal tumors, and germ cell tumors). Ovarian cancer can include fallopian tube cancer or primary peritoneal carcinoma. The cancer can be characterized by high expression of folic acid receptor α (FOLR1) and can be, for example, ovarian cancer. The cancer can be treated by accumulating cytotoxic T cells in folic acid receptor positive (FR+) tumor cells. In some embodiments, the present invention provides a method of treating a genetic disease, AIDS, or diabetes by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. In some embodiments, the anti-CD3 antibody or treatment is a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is optionally conjugated with two folic acid molecules and two PEGylated molecules and is a bispecific antibody comprising non-naturally encoded amino acids site-specifically incorporated. In further embodiments, in the anti-CD3 Fab antibody, two folic acid molecules and two PEGylated molecules are conjugated via the side chains of non-natural amino acids.

[0253] The present invention provides an anti-CD3 antibody for use in treating a disease or medical condition in cells that highly express a folate receptor. The anti-CD3 antibody of the present invention is used for treating cancer, and examples of the cancer include, but are not limited to, ovarian cancer (including epithelial tumors, stromal tumors, and germ cell tumors). Ovarian cancer may include fallopian tube cancer or primary peritoneal carcinoma. The cancer is characterized by high expression of folate receptor α (FOLR1) and may be, for example, ovarian cancer. The cancer can be treated by accumulating cytotoxic T cells in folate receptor-positive (FR+) tumor cells. The anti-CD3 antibody of the present invention is used for treating genetic diseases, AIDS, diabetes (not limited thereto). The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating a disease or medical condition in cells that highly express a folate receptor. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating cancer, and examples of the cancer include, but are not limited to, ovarian cancer (including epithelial tumors, stromal tumors, and germ cell tumors). Ovarian cancer may include fallopian tube cancer or primary peritoneal carcinoma. The cancer is characterized by high expression of folate receptor α (FOLR1) and may be, for example, ovarian cancer. The cancer can be treated by accumulating cytotoxic T cells in folate receptor-positive (FR+) tumor cells. The anti-CD3 antibody of the present invention can be used in the manufacture of a medicament for treating genetic diseases, AIDS, diabetes (not limited thereto).

[0254] In some embodiments, the condition to be treated is cancer. Examples of cancer include, but are not limited to, breast cancer, brain tumor, pancreatic cancer, skin cancer, lung cancer, liver cancer, gallbladder cancer, colon cancer, ovarian cancer, prostate cancer, uterine cancer, bone cancer, and blood cancer (leukemia), or cancers, diseases, or conditions associated with any of these cancers. Carcinomas are cancers that occur in epithelial cells, which cover the body's surface, produce hormones, and form glands. Non-limiting examples of carcinomas include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain tumor, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, fallopian tube cancer, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, melanoma of the skin or uvea, anal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, renal pelvis cancer, ureteral cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasms, CNS primary lymphoma, brainstem glioma, and spinal cord tumors. In some cases, it is skin cancer such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic keratosis (actinic keratosis). In some embodiments, the cancer is any cancer having highly expressed folate receptor α or β. In some embodiments, the condition to be treated is a disease or condition. The disease or condition can be a pathogenic infection. The pathogenic infection can be a bacterial infection. The pathogenic infection can be a viral infection. The disease or condition can be an inflammatory disease. The disease or condition can be an autoimmune disease. The autoimmune disease can be diabetes. The disease or condition can be cancer. In some embodiments, the disease or condition is any disease or condition having highly expressed folate receptor α or β. The disease or condition can be a pathogenic infection. The biologically active molecule can interact with cell surface molecules on infected cells. The biologically active molecule can interact with molecules on bacteria, viruses, or parasites. The pathogenic infection can be induced by one or more pathogens. In one example, the pathogen is a bacterium, fungus, virus, or protist.Representative pathogens include, but are not limited to, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia coli, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia. The pathogen can be a virus. Examples of viruses include, but are not limited to, adenovirus, coxsackievirus, Epstein - Barr virus, hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C), herpes simplex virus (types 1 and 2), cytomegalovirus, herpesvirus, HIV, influenza virus, measles virus, mumps virus, papillomavirus, parainfluenza virus, poliovirus, RSV, rubella virus, and varicella - zoster virus. Examples of diseases or conditions induced by viruses include, but are not limited to, colds, influenza, hepatitis, AIDS, chickenpox, rubella, mumps, measles, warts, and polio. The disease or condition can be an autoimmune disease or an autoimmune - related disease. An autoimmune disease is a malfunction of the body's immune system that can induce the body to attack its own tissues.Examples of autoimmune and autoimmune-related diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, Behçet's disease, celiac sprue, Crohn's disease, dermatomyositis, eosinophilic fasciitis, erythema nodosum, giant cell arteritis (temporal arteritis), Goodpasture syndrome, Graves' disease, Hashimoto's disease, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, Kawasaki disease, Lambert-Eaton syndrome, lupus (SLE), mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus, polyarteritis nodosa, type I and type II and type III autoimmune polyendocrine syndromes, polymyalgia rheumatica, polymyositis, psoriasis, psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0255] The disease or condition can be an inflammatory disease. Examples of inflammatory diseases include, but are not limited to, cellulitis, amyloidosis, vasculitis, ankylosing spondylitis, avascular necrosis, Graves' disease, Bell's palsy, synovitis, carpal tunnel syndrome, celiac disease, cholangitis, chondromalacia patellae, chronic hepatitis, chronic fatigue syndrome, Cogan's syndrome, congenital hip dysplasia, costochondritis, Crohn's disease, cystic fibrosis, De Quervain's tenosynovitis, diabetes-related arthritis, diffuse idiopathic skeletal hyperostosis, discoid lupus, Ehlers-Danlos syndrome, familial Mediterranean fever, myositis, fibrositis / fibromyalgia, frozen shoulder, ganglion cyst, giant cell arteritis, gout, Graves' disease, HIV-related rheumatic disease syndrome, hyperparathyroidism-related arthritis, infectious arthritis, inflammatory bowel syndrome / irritable bowel syndrome, juvenile rheumatoid arthritis, Lyme disease, Marfan syndrome, Mikulicz's disease, mixed connective tissue disease, multiple sclerosis, myofascial pain syndrome, osteoarthritis, osteomalacia, osteoporosis and corticosteroid-induced osteoporosis, Paget's disease, relapsing rheumatism, Parkinson's disease, plasma cell disease, polymyalgia rheumatica, polymyositis, pseudogout, psoriatic arthritis, Raynaud's phenomenon / syndrome, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, sciatica (lumbar radiculopathy), scleroderma, scurvy, sickle cell arthritis, Sjogren's syndrome, spinal stenosis, spondylolisthesis, Still's disease, systemic lupus erythematosus, Takayasu (pulseless) disease, tendinitis, tennis elbow / golfer's elbow, thyroid-related arthritis, trigger finger, ulcerative colitis, Wegener's granulomatosis, and Whipple's disease.

[0256] For a human patient suffering from a disorder that can be affected by the use of an anti-CD3 antibody agonist or antagonist, a pharmaceutical composition containing an anti-CD3 antibody can be formulated to be effective when administered by various means. Effects of the anti-CD3 antibody agonist or antagonist include, but are not limited to, anti-proliferation, anti-inflammation, anti-virus, etc. This effect may be directed against the condition or the disease itself, or against a part of the condition or disease. The average amount of the anti-CD3 antibody can vary. In particular, it should be varied based on the recommendation and prescription of a qualified physician. The exact amount of the anti-CD3 antibody is a matter of preference if the prerequisite factors such as the exact type of the condition to be treated, the condition of the patient to be treated, and other components in the composition are satisfied. The present invention also provides the administration of a therapeutically effective amount of other active ingredients (including, but not limited to, chemotherapeutic agents or immunotherapeutic agents for anti-cancer). A person skilled in the art can easily determine the dosage based on the treatment using the anti-CD3 antibody.

[0257] 〔Example〕 The following examples are provided for illustrative purposes and do not limit the invention according to the claims.

[0258] 〔Example 1〕 When incorporating amino acids not naturally encoded in an anti-CD3 antibody, a set of criteria for selecting suitable sites will be described. What is shown in this example is a method for selecting a suitable site in the antigen-binding polypeptide CD3 for introducing amino acids not naturally encoded. The three-dimensional structure composed of two anti-CD3 antibody molecules, or the secondary, tertiary, or quaternary structure of the anti-CD3 antibody, is utilized to select a suitable site.

[0259] The following criteria are used to evaluate each position of the anti-CD3 antibody in introducing non-naturally encoded amino acids. That is, the residue must not (a) interfere with the binding of the anti-CD3 antibody based on the structural analysis of the three-dimensional structure, nor based on the secondary, tertiary, or quaternary structure of the anti-CD3 antibody; (b) be affected by scanning mutagenesis using alanine or a homolog; (c) not be exposed on the surface, and the van der Waals interaction or hydrogen bond interaction with surrounding residues must be minimized; (d) may be at one or more of the exposed surfaces of the anti-CD3 antibody; (e) may be at one or more sites of the anti-CD3 antibody located proximal to a second anti-CD3 antibody, or another molecule or fragment thereof; (f) in the anti-CD3 antibody variant, must be either deleted or variable; (g) may be conservatively changed as a result of substitution with a non-naturally encoded amino acid; (h) may regulate the conformation of the anti-CD3 antibody itself, or a dimer or multimer containing one or more anti-CD3 antibodies, by desirably changing the overall flexibility or rigidity; (i) may be in either highly flexible regions or structurally rigid regions; (j) may or may not be in the complementarity determining region (CDR). Further calculations are performed on the anti-CD3 antibody molecule using the Cx program (Pintar et al. Bioinformatics, 18, pp 980) to evaluate the degree of protrusion for each atom in the protein. As a result, in some embodiments, one or more sites of the anti-CD3 antibody are substituted with non-naturally encoded amino acids.

[0260] Example 2 The expression of anti-CD3 antibodies having non-naturally encoded amino acids in Escherichia coli will be described in detail. An anti-CD3 antibody having a non-naturally encoded amino acid was expressed using a translational system equipped with an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS). The O-RS preferentially aminoacylates a non-naturally encoded amino acid and the O-tRNA. Next, the translational system inserts the non-naturally encoded amino acid into the anti-CD3 antibody corresponding to the encoded selected codon.

[0261] By transforming Escherichia coli using a plasmid containing a modified anti-CD3 antibody gene and an orthogonal aminoacyl tRNA synthetase / tRNA pair (specific for the desired non-naturally encoded amino acid), non-naturally encoded amino acids can be site-specifically incorporated into the anti-CD3 antibody. The transformed Escherichia coli is cultured at 37 °C in a medium containing 0.01 to 100 mM of a specific non-naturally encoded amino acid to express the modified anti-CD3 antibody with high accuracy and efficiency. An anti-CD3 antibody having a non-naturally encoded amino acid is produced in an Escherichia coli host cell. The product is obtained as a soluble protein in the periplasm. Methods for purifying the anti-CD3 antibody are well known and can be confirmed by SDS-PAGE, Western blot analysis, or electrospray ionization-ion trap mass spectrometry, etc.

[0262] [Expression / Suppression] Suppression by non-naturally encoded amino acid (para-acetylphenylalanine (pAF)): Suppress the amber mutation of E. coli according to a known standard protocol. Briefly, the following operations were performed to suppress the antibody fragment (Fab) in the periplasm of E. coli. First, an expression vector construct was transformed into E. coli host cells with a plasmid encoding an orthogonal tRNA synthetase (e.g., the orthogonal tyrosyl-tRNA synthetase (MjTyrRS) from M. jannaschii). After one night, the bacterial culture was diluted 1:100 with shaking in a flask containing LB (Luria-Bertani) medium or Superbroth. Then, it was cultured at 37 °C until the OD reached about 0.8. Para-acetylphenylalanine (pAF) was added to a final concentration of 4 mM to suppress the amber codon and induce the expression of Fab. The culture was incubated overnight at 25 °C.

[0263] Suppression by non-naturally encoded amino acid derivatives: The amber mutation is suppressed by a derivative of a non-naturally encoded amino acid (e.g., pAF(aa9.2)) in the same manner as described above, except that an orthogonal synthetic enzyme specific for this amino acid (e.g., the tyrosyl-tRNA synthetase (MjTyrRS) from M. jannaschii) is used. For example, during induction, it is suppressed by adding 4 mM of aa9.2.

[0264] Cells are harvested by centrifugation and resuspended in periplasmic release buffer (50 mM NaPO4, 20% sucrose, 1 mM EDTA; pH 8.0) supplemented with 100 μg / ml lysozyme. Then, they are incubated on ice for 30 minutes. After centrifugation, the antibody fragments in the supernatant are immobilized onto ProBind beads (Invitrogen; Carlsbad, CA) using the His-tag of the antibody fragments. After washing the beads well with binding buffer, the bound fragments are eluted from the beads with 0.5 M imidazole. The purified fragments are dialyzed in storage buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 5% sucrose; pH 7.8). For small-scale analysis of Fab fragments expressed in the periplasm, Escherichia coli in 15 ml of medium is harvested by centrifugation. Then, it is resuspended in 1 ml of lysis buffer (B-PER, Pierce Biotechnology; Rockford, IL) supplemented with 10 μg / ml DNase. The mixture is incubated at 37 °C for 30 minutes and diluted 1× with Protein Loading buffer (Invitrogen; Carlsbad, CA) and analyzed by SDS-PAGE.

[0265] Example 3 Design and construction of the humanized anti-CD3 gene in the pFUSE vector - The mouse monoclonal anti-CD3 antibody SP34 (Harvard BIDMC) was humanized. Based on in silico analysis and design, three variable heavy chains (vH1.0, vH1.1 & vH1.2) and three variable light chain genes (vL1.0, vL1.1 & vL1.2) (Genewiz, South Plainfield, NJ) containing various mouse framework revertant mutations in addition to the mouse CDR sequences to the selected human framework scaffolds were synthesized. Table 2 lists the mouse framework residues (revertant mutations) along with Kabat numbering, which were retained in the three variable heavy chains (vH) and three variable light chains (vL) of the nine humanized anti-CD3 Fabs described in Figure 1. Amino acid residues reverted to the human framework sequence are shown in bold.

[0266]

Table 2

[0267] As shown in Table 2, five mouse back mutations (V36, G46, G49, G57, and V58) were present in the variable light chain, and four mouse back mutations (N30, A49, I77, and V93) were present in the variable heavy chain. Six short synthetic variable genes shown in Table 2 were cloned into InvivoGen's heavy chain (HC) and light chain (LC) expression vectors (pFUSE-CHIg-HG1 and pFUSE-CLig-hk, respectively) to obtain a plasmid expressing the humanized anti-CD3 SP34 antibody. As shown in Figure 1 and Table 2, the variable light chain vL1.0 with all five mouse back mutations converted to human residues lost its binding to any combination of variable heavy chains (vH).

[0268] 〔Example 4〕 Expression of humanized anti-CD3 antibody in the HEK293 transient system: The humanized SP34 antibody described in the above example was transiently expressed in HEK293 cells by combining the plasmid containing each vH gene with the plasmid containing each vL gene (co-transfection). The protein concentration in the cell culture medium was measured from each co-transfection experiment and used directly in the PBMC-based CD3 binding assay without further purification. The controls used were the chimeric SP34 construct (positive control) and the irrelevant PSMA antibody construct (negative control). Humanized anti-CD3 antibodies without non-natural amino acid incorporation and with or without the HC-DKTHT extension were also expressed and characterized in HEK293 cells (Table 3). These antibodies were used for screening for low-affinity Fab mutants. Table 3 shows the novel wild-type (WT) amino acid sequences of the humanized anti-CD3 heavy chain (vH+CH1) and light chain (vL+CL) sequences used in various combinations to generate the Fab WT sequences described herein. The WT amino acid sequence of the humanized anti-CD3 heavy chain (vH+CH1-DKTHT) sequence is also shown in Table 3.

[0269]

Table 3

[0270] 〔Example 5〕 Test for CD3 binding with activated human and cyno PBMC: To distinguish the generated humanized anti-CD3 antibodies, binding to human CD3 was tested using activated human PBMC (n = 2). Activation of PBMC and subsequent fluorescence-based CD3 binding assays were performed as previously described (see, for example, Angew Chem Int Ed Engl, 52(46):12101 - 12104, 2013). As shown in Figure 1, three antibodies with the vL1.0 light chain lost binding to human CD3 in combination with any vH heavy chain. Binding of the remaining six humanized antibodies to both human and cyno CD3 was titrated using activated human and cyno PBMC, respectively. As shown in Figures 2A - 2F, all six humanized anti-CD3 antibodies (engineered from 3 vH heavy chains using the 2 vL light chain combinations shown in Table 3) retained equivalent binding to both human and cyno CD3. The six humanized anti-CD3 antibodies were obtained as follows: Fab1, Fab2, and Fab3 wild types were generated by combinations of LC ((vL1.2 + CL); (SEQ ID NO: 7)) and HC ((vH1.2 + CH1); (SEQ ID NO: 1)), ((vH1.1 + CH1); (SEQ ID NO: 2)), and ((vH1.0 + CH1); (SEQ ID NO: 3)), respectively. Fab4, Fab5, and Fab6 wild types were generated by combinations of LC ((vL1.1 + CL); (SEQ ID NO: 8)) and HC (((vH1.2 + CH1); (SEQ ID NO: 1)), ((vH1.1 + CH1); (SEQ ID NO: 2)), and ((vH1.0 + CH1); (SEQ ID NO: 3)), respectively.

[0271] Regarding the binding to both CD3 substrates, since no significant differences were shown among the six humanized antibodies, Fab1 (the combination of vH1.2 + vL1.2) was selected to further demonstrate an important aspect of the present invention. For example, Fab1 was used for expression in E. coli for further optimization using the unique unnatural amino acid (UAA) incorporation technology by the previously described orthogonal amber suppression system (see, for example, WO2017 / 079272, WO2012 / 166560, and WO2013 / 192360). The list of mouse framework revertant mutations required to maintain the binding activity in these six Fabs is shown in Table 4, which provides the list of mouse framework residues (revertant mutations) retained in the six humanized anti-CD3 Fabs described in FIGS. 2A - 2F together with Kabat numbering. Amino acid residues reverted to the human framework sequence are shown in bold. The combination of vL1.2 and vH1.2 variable chains resulting in Fab1 (underlined) was selected as a lead for further modification.

[0272]

Table 4

[0273] 〔Example 6〕 Cloning of the synthetic Fab gene into an E. coli expression vector: The synthetic Fab gene was cloned into a unique standard E. coli expression vector using the Gibson Assembly cloning kit (New England Biolabs). After sequence verification of each expression plasmid, each plasmid was transformed into the standard E. coli production host W3110B60 strain, and a single colony isolated for each plasmid was purified to prepare glycerol vials. The glycerol vials served as production clones for the E. coli fermentation of these Fab molecules. The amino acid sequences of the four heavy chains and three light chains used when manipulating these Fabs are shown in Table 5 as SEQ ID NOs: 10 - 20.

[0274]

Table 5

[0275] JPEG2025098156000027.jpg63164

[0276] Three light chain (LL157pAF, LK172pAF, and LS205pAF) pAF variants (SEQ ID NOs: 18-20), as well as three double pAF variants (((HK129pAF+LL157pAF); (SEQ ID NOs: 16 and 18)); (HK129pAF+LK172pAF); (SEQ ID NOs: 16 and 19)), and ((HK129pAF+LS205pAF) (SEQ ID NOs: 16 and 20)) were designed. SEQ ID NOs: 10-13 represent heavy chain pAF variants that do not contain the 5-aa heavy chain C-terminal extension - DKTHT.

[0277] E. coli fermentation: The fermentation process for the production of anti-CD3 Fab-pAF consists of two stages: (i) inoculum preparation and (ii) fermentor production. The inoculum material is started from a single glycerol vial, thawed, diluted 1:1000 (v / v) into 50 mL of defined seed medium in a 250 mL baffled Erlenmeyer flask, and incubated at 37 °C and 250 rpm. Before use, the fermentor is cleaned and autoclaved. A specific amount of basal medium is added to the fermentor and steam sterilized. A specific amount of kanamycin sulfate solution, feed medium, and P2000 antifoam agent are added to the basal medium before inoculation. All solutions added to the fermentor after autoclaving are either 0.2 μm filtered or autoclaved before aseptic addition.

[0278] The production fermentor is inoculated at a target OD of 0.0004 by aseptically transferring the contents of the inoculum material. 600 After inoculation, the medium is sampled at appropriate intervals for OD 600It is determined. Temperature, pH and dissolved oxygen are monitored and controlled at specific set points of 37 °C, 7.0 and ≥ 30% respectively. The pH is controlled by the addition of ammonium hydroxide solution or sulfuric acid. Dissolved oxygen is controlled by changing the stirring speed and by increasing the composition of oxygen in the sparged air / oxygen mixture. An antifoaming agent is added during the fermentation process to control foaming.

[0279] When the cell density reaches > 25 OD 600 a bolus of the feed medium is added. When the cell density reaches > 50 OD 600 a feed medium is added at a constant flow rate of 0.094 mL / L starting volume per minute for 32 hours until it decreases to 0.052 mL / L starting volume per minute and the fermentation ends. Immediately after the start of the feed, a specific amount of a solution of a non-naturally encoded amino acid (e.g., pAF) is added aseptically to incorporate the non-natural amino acid into the protein amino acid sequence. At the same time, the temperature is shifted from 37 °C used during growth to 27 °C for production. Production is controlled by the phoA promoter and is initiated when the phosphate level in the medium is depleted. Recovery is initiated approximately 48 hours after induction.

[0280] [Example 7] Purification, and complexation with folic acid and PEG-folic acid: The anti-CD3 Fab of the present invention is produced in E. coli cells and recovered from the whole cell lysate (WCL) supernatant. Cell lysis is carried out at 4 °C. The cells are lysed in a volume equal to the initial fermentation volume in 100 mM acetic acid, 100 mM NaCl, 1 mM EDTA, pH 3.5 to obtain a lysed product with a pH of 4.1 - 4.2.

[0281] After dissolution, the WCL is centrifuged at 15,900×g for 30 minutes at 4°C and filtered (0.8 / 0.2 micron) to remove precipitated proteins and cell debris. Subsequently, Capto S cation exchange chromatography is used to capture anti-CD3 Fab from the E. coli WCL supernatant. Following the Capto S column, butyl HP hydrophobic interaction chromatography (HIC) is used as a polishing column to isolate the complete anti-CD3 Fab from product-related impurities present in the Capto S elution pool. The butyl HP elution pool containing the complete anti-CD3 Fab is then buffer-exchanged into 50 mM acetate, 5% trehalose, pH 4.0 and concentrated in preparation for conjugation with folic acid or PEG-folic acid. This step is performed at 4°C using an Amicon Ultracel 10K regenerated cellulose (15 mL) centrifuge device.

[0282] After buffer exchange and concentration into 50 mM acetic acid, 5% trehalose, pH 4, the anti-CD3 Fab is conjugated with folic acid or PEG-folic acid to target the folate receptor on cancer cells. The conjugation reaction is carried out at 28°C, pH 4 for 24 - 48 hours. After conjugation with folic acid, the anti-CD3 Fab-folic acid is buffer-exchanged into the preparation buffer, (50 mM histidine, 100 mM sodium chloride, 5% trehalose, pH 6.0). This step is performed at 4°C using an Amicon Ultracel 10K regenerated cellulose (15 mL) centrifuge device. After conjugation with PEG-folic acid, Toyo SP 5PW cation exchange chromatography is used to separate uncomplexed, single, and double complexed anti-CD3 Fab-PEG-folic acid.

[0283] CD3 Fab folic acid-5KPEG, CD3 Fab folic acid-10KPEG, CD3 Fab folic acid-20KPEG, CD3 Fab folic acid-(5K)2PEG, and CD3 Fab folic acid-(10K)2PEG compounds were prepared. The desired folic acid-PEG (5K, 10K, 20K, 5K2, or 10K2PEG) was added to CD3 Fab in buffer (50 mM acetic acid, 5% trehalose, pH 4) at 28 °C. After 1 hour, the mixture was purified by cation exchange chromatography (Toyo SP 5PW) and adjusted with buffer of 50 mM histidine, 100 mM NaCl, 5% trehalose, pH 6.0 by using a centrifugal filter (c / o 10K) to obtain the desired CD3 Fab-folic acid-PEGylated composition. The structure, chemistry, and complexation of the CD3 Fab folic acid complex are described herein and shown in FIGS. 12A-12D.

[0284] Furthermore, CD3 Fab folic acid-PEGylated C-terminal complexes were prepared. EDTA (6.7 μL, 0.5 M, pH 8) and DTT (0.4 mg) were added to CD3 folic acid (8.0 mg) in PBS (2.0 mL), and the solution was incubated at 37 °C for 30 minutes. The mixture was purified by a desalting column with 5 mmol of EDTA in PBS eluent. Mal-PEG was added to the mixture at various concentrations (4.2 mg of 5K, 8.2 mg of 10K, or 16 mg of 20K) at room temperature. After 2 hours, the mixture was purified by Toyo SP 5PW cation exchange chromatography to obtain CD3 Fab folic acid-(PEG5K)2 C-terminal complex, CD3 Fab folic acid-(PEG10K)2 C-terminal complex, and CD3 Fab folic acid-(PEG20K)2 C-terminal complex. The structure, chemistry, and complexation of the C-terminal PEG complex are described herein and shown in FIGS. 12E-12F.

[0285] [[Example 8]] In Vitro Binding and Killing Assays: Purified humanized anti-CD3 Fabs with folic acid conjugated at various heavy chain sites (HA114, HS115, HK129, HT160) were first tested for binding to both human and cyno CD3, together with the corresponding unbound proteins having pAF at these positions, as well as the WT protein as a control. Table 6 shows the EC 50 of modified anti-CD3 Fab1 proteins purified from E. coli cells to activated human and cyno PBMC. As shown in Table 6, neither pAF at different sites nor conjugated folic acid significantly interfered with CD3 binding.

[0286] [Table 6]

[0287] The cytotoxicity of these folic acid-conjugated anti-CD3 Fabs was tested at an E:T = 10:1 ratio using activated human and cyno PBMC with SKOV-3 cells in the presence or absence of 50 nM serum folic acid (SFA). The cytotoxicity assay was performed as previously described (see, for example, Angew Chem Int Ed Engl, 52(46):12101-12104, 2013). Briefly, effector cells (either activated PBMC or non-activated PBMC) and target cells (SKOV-3, KB, etc.) at various E:T ratios were co-incubated overnight or as indicated in a U-bottom 96-well plate with anti-CD3Fab-folic acid at various concentrations. The amount of LDH released into the medium was used as an indicator of cytotoxicity and measured using Promega's non-radioactive cytotoxicity assay kit according to the manufacturer's instructions.

[0288] As shown in Table 7, there was no significant difference among the conjugated sites regarding the killing of EC 50 . However, in the presence of 50 nM SFA, due to competitive inhibition by free SFA, a decrease of approximately 15 - 60-fold in cytotoxic EC 50 was observed compared to the case without SFA. Also, cyno EC 50 was 50was approximately 10-fold higher in each variant than

[0289]

Table 7

[0290] 〔Example 9〕 Cytotoxicity of anti-CD3-folate variants against various FOLRα tumor cell lines having various folate receptor (FRα) levels: The activities of three different anti-CD3Fab-folate conjugates were tested at the HA114, HS115, and HK129 sites by an in vitro cytotoxicity assay using six different cell lines having various levels of FRα overexpression on the cell surface (Table 8). The number of FRα varied from 5,700 per cell in the alveolar basal epithelial carcinoma A549 cell line to 1,630,000 per cell in the hypopharyngeal carcinoma cell line. Cells were co-cultured with activated human peripheral blood mononuclear cells (PBMCs; target: effector cell ratio 1:10) and treated with various concentrations of anti-CD3Fab-folate in the presence of 50 nM SFA. Cytotoxicity was quantified by measuring the LDH levels released from lysed cells using a cytotoxicity assay based on CellTiter-Glo and FACS.

[0291]

Table 8

[0292] Anti-CD3Fab-folate conjugates at all three different sites of conjugation showed efficient killing of all five cell lines having more than 10K FRα. However, no killing was observed in the A549 cell line having any site conjugate with 5,700 FRα. There appears to be a threshold of approximately 10,000 FRα for efficient killing, but the killing activity appears to be independent of the number of FRα above the threshold of 10K. The in vitro killing EC 50 did not vary significantly at the folate conjugate site in the antibody, so the HK129 position was selected as the folate conjugate site for further experiments.

[0293] 〔Example 10〕 This example demonstrates the effect of various anti-CD3 Fab1-HK129-pAF molecules on the in vitro binding affinity and cytotoxic activity against either the addition of folic acid-PEG conjugate or a second folic acid (difolic acid) at a single site.

[0294] Effect of folic acid-PEG conjugate: Table 9 shows the effect on binding, and Table 10 shows the cytotoxicity against the conjugate of folic acid with a linear PEG molecule of either 5K or 20K using a bifunctional linker as described herein. A slight decrease in binding (1.7 - 8.4-fold) was observed for both activated human and cyno PBMCs depending on the 5K or 20K PEG size. However, the cytotoxic activity decreased dramatically (3.7 - 5.6-fold vs 50 - 58-fold) with 20K PEG compared to 5K PEG for both human and cyno PBMCs. Based on this experiment, 5K PEG was used instead of 20K PEG for half-life extension.

[0295]

Table 9

[0296]

Table 10

[0297] Effect of difolic acid conjugate: Table 11 shows the effect on the in vitro binding of a second folic acid conjugation at two light chain sites (LL157 and LK172) combined with the heavy chain HK129 site, and Table 12 shows the cytotoxicity. As expected, a slight increase in both binding affinity and cytotoxic activity was observed for the difolic acid variant over the HK129 single folic acid molecule. A slight increase in the cytotoxicity of the double versus single folic acid molecule was also evident in the presence or absence of 50 nM SFA. Based on this experiment, the LL157 site was used in combination with the HK129 site for future studies over the LK172 site.

[0298]

Table 11

[0299]

Table 12

[0300] Summary of binding affinity and in vitro cytotoxicity: Table 13 summarizes the effects of PEGylation and second folic acid conjugation on the binding and cytotoxic activities of various anti-CD3 Fabs. A slight decrease in the binding affinity for both CD3 and FRα results in a significant decrease in efficacy, which correlates with the size of the PEG. The conjugate of the two folic acid molecules increased the affinity for FRα and thereby increased the cytotoxic efficacy, despite some decrease in the CD3 binding affinity. Despite the decrease in affinity for both targets due to PEG conjugation, the in vitro killing EC50 of all CD3-Fab-folic acid molecules maintained high efficacy between 37 and 335 pM.

[0301]

Table 13

[0302] 〔Example 11〕 Effect of various effector-to-target (E:T) cell ratios on the cytotoxicity of SKOV-3 cells by anti-CD3 Fab1 molecules: To test the effect of the E:T ratio on the cytotoxicity of SKOV-3 cells, cytotoxicity assays of three anti-CD3 Fab1 molecules were performed at E:T ratios of 10:1, 5:1, 1:1, 1:5, and 1:10 in the presence of 50 nM SFA. As expected, as shown in Table 14, the E:T ratio correlated with the in vitro killing efficacy. At E:T = 1:1, a two-fold increase in EC 50 was observed for the folic acid molecule, while an eight-fold decrease was seen for the 5KPEG-folic acid molecule. All three molecules remained highly effective with EC 50 between 1.69 and 175 pM within the E:T ratio of 10:1 to 1:10.

[0303]

Table 14

[0304] 〔Example 12〕 PEGylation of anti-CD3 Fab1 molecules to extend plasma half-life: In rats, the pharmacokinetic properties of anti-CD3 Fab1-folate conjugates were investigated with and without PEG conjugation. Male Sprague Dawley rats (about 7 weeks old) were used. On the day of administration, the body weights of each animal were measured. Samples of non-conjugated anti-CD3 antibody or conjugated anti-CD3 antibody at 1 mg per kg body weight were intravenously injected into groups of three rats each via the tail vein. At different time points after injection, 500 μl of blood was collected from each rat under anesthesia with CO2. The blood samples were stored at room temperature for 1.5 hours and then serum was separated by centrifugation (5 minutes at 18,000×g at 4°C). Serum samples were stored at -80°C until the day of analysis. After thawing the samples on ice, the amount of active anti-CD3 antibody in the serum samples was determined by an anti-CD3 antibody in vitro activity assay.

[0305] As shown in Figure 3, serum concentrations rapidly decreased at the same rate with a serum half-life of less than 1 hour for both anti-CD3 Fab1-folate and the corresponding non-conjugated anti-CD3 Fab1. In contrast, the serum half-lives of the two PEG-conjugated anti-CD3 Fab1 were significantly extended to 6.1 hours and 10.6 hours for 5KPEG and 25KPEG, respectively. Table 15 shows various PK parameters after IV administration of various anti-CD3 Fab1 molecules in rats. This data shows that 25KPEG-difolate extends the T1 / 2 and significantly increases the AUC (13.2-fold for Fab1-folate and 3.9-fold for Fab1-5KPEG-folate) (Table 15).

[0306]

Table 15

[0307] [Example 13] This example demonstrates the generation and screening of anti-CD3 low-affinity antibody variants in HEK293 cells.

[0308] Mouse back mutations and germline mutations: Mouse framework residues (back mutations) for both the vH and vL sequences obtained during humanization of the anti-CD3 antibody were reverted to human germline residues all at once, and their effects in antigen binding (deconvolution) were examined by seeing their effects. For the vH sequence, four mouse framework residues (back mutations) at Kabat positions 30, 49, 77, and 93 were predicted to play important roles in antigen binding (Tables 2-3 and 16-17). Similarly, for the vL sequence, five mouse framework residues (back mutations) at positions 36, 46, 49, 57, and 58 were also predicted to play important roles in antigen binding.

[0309] [Table 16]

[0310] [Table 17]

[0311] To evaluate the effects on antigen binding (deconvolution), four new vL (vL2.1-vL2.4) and three new vH (vH2.1-vH2.3) plasmids (round 1 plasmids in Tables 16-17) were generated. These vH and vL plasmids were used in co-transfection assays using HEK293 cells together with the original vH and vL plasmids described in the above examples (round 0 plasmids in Tables 16-17). A total of 35 transient transfections were performed in round 1 screening, and the cell culture supernatants were used directly to screen for binding as described in the following and above examples.

[0312] Based on the screening results of Round 1, three additional vH plasmids (vH2.4 - 2.6) (Round 2 plasmids in Tables 16 - 17) were constructed to evaluate the additive effects of individual reverted mutations. Similarly, a new vL (vL2.5) plasmid was constructed by combining two mouse reverted mutations. Furthermore, mouse germline cell mutations found within the vH and vL CDRs were analyzed to confer decreased binding in this round. For this purpose, three new vH plasmids (vH3.1 - 3.3) (Round 2 plasmids in Tables 16 - 17) were generated by changing the amino acids at Kabat position N35 of HC - CDR1 and Y52c of HC - CDR2. Similarly, a vL plasmid (vL3.1) (Round 2 plasmids in Tables 16 - 17) was constructed by changing the amino acid at Kabat position K53 of LC - CDR2. The Round 2 screening experiment was performed in a total of 43 transfections by selectively combining various vL / vH plasmid pairs.

[0313] Screening for human and cyno CD3 binding: To test for binding to human CD3 at the screening stage, HEK293 cell supernatants were used directly. In the Round 1 experiment, initially 35 clones were tested for binding to human CD3 at three different protein concentrations, and 13 were selected for detailed binding assays for both human and cyno CD3. For each clone, 11 - point binding titration curves were generated for both human (Figure 4A) and cyno CD3 (Figure 4B). Based on this data, Fab7 - 10 were selected as low - affinity candidates for further evaluation (Figure 4A - 4B). Similarly, 43 clones were screened in the Round 2 experiment. After the two - step screening as described above for Round 1, 11 low - affinity variants were obtained, four of which are shown in Figures 5A - 5B.

[0314] Next, these 15 low-affinity anti-CD3 Fab variants (Fab7-10 from round 1 and Fab11-21 from round 2) were transferred to an E. coli expression system for non-natural amino acid (UAA) incorporation and further tested as described in the following examples. Figure 6A shows the Fab-folate complex of the same wild-type Fab as shown in Figure 4A. A Fab-folate complex representative of the wild-type Fab obtained from round 2 screening is shown in Figure 6B. Table 18 shows a list of the low-affinity mutations and their HC, LC, and Fab IDs. As shown, both mouse framework revertant mutations and germline CDR mutations played important roles in conferring a decrease in binding to both human and cyno CD3. In the variable light chain (vL), five mouse revertant residues at Kabat positions V36, G46, G49, G57, and V58 of LC-CDR2 and one mouse germline residue at Kabat position K53 were involved in the decrease in binding. In the variable heavy chain (vH), four mouse revertant residues at Kabat positions N30, A49, I77, and V93 of HC-CDR1, and two mouse germline residues at Kabat position N35 of HC-CDR1 and Y52c of HC-CDR2 were involved in the decrease in binding.

[0315]

Table 18

[0316]

Table 19

[0317] JPEG2025098156000042.jpg217164

[0318] SEQ ID NOs: 21-29 are 5-aa used for screening of low-affinity variant Fabs Represents the amino acid sequence of the heavy chain having a C-terminal extension - DKTHT. SEQ ID NOs: 30 and 38 represent these humanized heavy chain amino acid sequences without a 5-aa heavy chain C-terminal extension. SEQ ID NO: 39 represents the amino acid sequence of the light chain used for screening low-affinity mutant Fabs in HEK293 cells.

[0319] [Example 14] This example demonstrates the construction, expression, purification, and testing of low-affinity humanized anti-CD3 Fab mutants produced from E. coli cells using the heavy chain HK129 amber mutation.

[0320] Cloning into the E. coli expression vector: Synthetic genes were designed for all low-affinity mutants (SEQ ID NOs: 40 - 62) disclosed in Table 20 using the STII-LC-spacer-STII-HC expression cassette structure with an amber TAG stop codon inserted at the heavy chain HK129 position, and cloned into a unique E. coli expression vector as described in the above examples. The amino acid sequences of both the heavy and light chains used to modify these Fabs are shown as SEQ ID NOs: 40 - 62. SEQ ID NOs: 40 - 48 represent humanized heavy chain HK129pAF mutants with a 5-aa heavy chain C-terminal extension - DKTHT used in the tests. SEQ ID NOs: 49 and 57 represent these humanized heavy chain HK129pAF mutants without a 5-aa heavy chain C-terminal extension - DKTHT. SEQ ID NOs: 58 - 62 represent the light chain sequences used in combination with the HK129pAF-DKTHT mutants of SEQ ID NOs: 40 - 48 expressed and further characterized in E. coli.

[0321] [Table 20]

[0322] JPEG2025098156000044.jpg217164

[0323] JPEG2025098156000045.jpg125164

[0324] Fermentation, purification, and folic acid conjugation: Escherichia coli fermentation, purification, folic acid conjugation, and post-conjugation purification were performed as described in the above examples.

[0325] Binding of folic acid-conjugated Fab to human and cyno CD3: Binding of the produced Escherichia coli and folic acid-conjugated low-affinity variants of humanized anti-CD3 Fab was performed as described in the above examples. Figures 6A - 6B show the binding affinity for human CD3 for two subsets of the low-affinity Fab variants of anti-CD3 Fab-HK129-folic acid, together with the positive control Fab1. Of the low-affinity Fab tested, 10 were unable to bind significantly to human CD3 even at the highest concentration tested (1000 nM). The binding EC 50 values of the remaining 4 Fabs (Fab7 - 10; listed in Table 18) varied from 23.4 nM to 83.6 nM compared to 2.31 nM for the control Fab1. Fab21 showed weak binding activity that did not saturate even at a concentration of 1000 nM. A very similar binding profile was observed for cyno CD3 binding as shown in Figures 7A - 7B.

[0326] Cytotoxicity assay of folic acid-conjugated Fab using human and cyno PBMC: The cytotoxicity assay of the low-affinity Fab was performed as described in the above examples. Figure 8 shows the cytotoxicity data of activated human PBMC with SKOV-3 cells. As shown, all 4 Fabs (Figures 6A - 6B) with 10 - 36-fold reduced binding affinity for human CD3 showed equivalent killing activity compared to the control Fab1 (Figure 8). Three Fabs (Fab11, 19, and 20) showed no cytotoxic activity (data not shown). The other 8 Fabs, all of which were unable to bind even at a concentration of 1000 nM, showed significant killing activity. A very similar cytotoxicity profile was observed with activated cyno PBMC (Figure 9).

[0327] Example 15 This example demonstrates the T cell activation and cytokine release assays of humanized anti-CD3 low-affinity antibody variants.

[0328] T cell activation / cytokine release assay: Purified human T cells and T cell-depleted human PBMCs were isolated from the same volume of whole blood using the EasySep Human T cell enrichment kit and the EasySep Human CD3 positive selection kit (STEMCELL Technologies Inc), respectively. The purity of the isolated T cells and accessory cells was confirmed by flow cytometry. To selectively monitor the activation of T cells in the presence of accessory cells, the purified T cells were labeled with the Cellvue Lavender cell labeling kit (eBioscience) according to the manufacturer's protocol before mixing with T cell-depleted human PBMCs. The resulting reconstituted PBMCs were incubated with target cells in the presence of an anti-CD3 Fab mutant. After 48 hours, the cells were labeled with APC-Cy7 conjugate anti-human CD25 (Biolegend) or PE conjugate anti-human CD69 (BD Biosciences) and analyzed by flow cytometry. The release of IFNγ and TNFα in the culture supernatant was measured by an enzyme-linked immunosorbent assay (ELISA) kit (R&D System).

[0329] As shown in FIGS. 10A-10B, the significant T cell activation (by CD25 and CD69 T cell markers) achieved in the presence of SKOV-3 cells is strongly dose-dependent on various low-affinity anti-CD3 Fab-HK129-folate molecules. Similar correlations were observed for IFNγ (FIGS. 11A-11B) and TNFα (FIGS. 11C-11D) in the absence and presence of SKOV-3 tumor cells, respectively.

[0330] Summary of Characterization of Low-Affinity Variants: Table 21 analyzes the data on binding and cytotoxicity (human and cyno CD3), as well as T cell activation (CD25 and CD69 markers) and cytokine release (IFNγ and TNFα) for 12 low-affinity anti-CD3 Fab variants with HK129-folate modification. As shown in the figure, there is a general correlation between the strength of CD3 binding, cytotoxic ability, T cell activation, and cytokine release.

[0331]

Table 21

[0332] Based on the dataset, three low-affinity variants, Fab9, Fab10, Fab21, were selected along with the parental molecule Fab1 for detailed in vitro characterization including in vivo testing in mice. Table 22 shows a comparison of these variants with respect to cytotoxicity and two cytokine productions. As shown, cytokine production has a much higher EC 50 than, much higher EC 50 for T cell activation and killing. Thus, it may be possible to identify an anti-CD3 Fab concentration range where cytokine release does not cause significant safety issues at that concentration, but efficacy and killing of T cell activation are not impaired. This approach of fine-tuning the anti-CD3 antibody affinity enables the separation of these two opposing events and may achieve a better safety profile without sacrificing efficacy.

[0333]

Table 22

[0334] 〔Example 16〕 In silico immunogenicity analysis of anti-CD3 Fab1, 9, and 10: To evaluate potential immunogenicity, the amino acid sequences of anti-CD3 Fab1, Fab9, and Fab10 were scanned in silico for the presence of putative human leukocyte antigen (HLA) class II-restricted epitopes, also known as T helper (Th)-cell epitopes, using the "HLA Class II - Global v4.0" settings (Lonza, UK) based on Lonza's Epibase platform. The HLA binding specificities of all possible 10-mer peptides derived from the target sequences were analyzed. Profiling was performed at the allotype level for 43 DRB1, 8 DRB3 / 4 / 5, 22 DQ, and 12 DP, i.e., 85 HLA class II allotypes in total. Peptides corresponding to self-peptides were treated separately as "germline-filtered" peptides. As a general overview of the results, Table 23 shows the number of strong binders corresponding to the DRB1, DRB3 / 4 / 5, DQ, and DP genes (number of epitopes). Similar to the case of the humoral response generated against the antigen, the observed Th cell activation / proliferation is generally interpreted from the perspective of DRB1 specificity. The results in Table 23 indicate that Fab 1, Fab9, and Fab10 correspond to strong potential DRB1 binders 13, 11, and 13, respectively, and Table 24 shows the DRB1 risk scores for each of these three Fabs in the global population comparable to that of the humanized treatment antibody. Among the three Fabs, Fab9 is the least immunogenic.

[0335] [Table 23]

[0336] [Table 24]

[0337] [Example 17] Design and synthesis of the bifunctional PEG-folate linker shown in FIGS. 12A - 12F. This example demonstrates the synthetic routes and structures of various PEG-folate linker compounds.

[0338]

Chem.

[0339] This example demonstrates the synthetic route for the synthesis of compound 10.

[0340]

Chem.

[0341] N 10 -Trifluoroacetylpteroic acid (2). 10 mL of anhydrous trifluoroacetic acid was added dropwise to 1.0 g of pteroic acid (1) in a round-bottom flask under nitrogen for 10 minutes. The reaction mixture was stirred at room temperature for 24 hours in the dark. The dark brown solution was filtered through a pad of celite and evaporated. The resulting viscous brown oil was triturated with ether, and the separated precipitate was collected by filtration, washed with ether, and dried overnight under vacuum to obtain the crude intermediate as a light brown powder. The crude acylated material was resuspended in anhydrous THF and treated with ice. The resulting mixture was stirred at room temperature for 10 hours; during this time, a light brown precipitate separated. The reaction mixture was diluted with ether, the precipitate was collected by filtration, washed with ether, and dried overnight under vacuum to obtain N10-trifluoroacetylpteroic acid (2) (1.45 g crude) as a light brown powder, which was used in the next reaction without further purification.

[0342] N 10 -Trifluoroacetylpteroic acid OSu ester (3). A solution of N 10 -Trifluoroacetylpteroic acid (2) in anhydrous DMSO was treated at room temperature all at once with N-hydroxysuccinimide (0.43 g), followed by EDCI-HCl (2.04 g). The resulting dark solution was stirred at ambient temperature for 24 hours and diluted with ice-cold water (40 mL). The separated fine brown precipitate was collected by centrifugation, washed with cold water, air-dried overnight, dried under vacuum for 1 day, and the compound (3) was obtained as a brown powder, MS (ESI) m / z 506 (M+H) + as obtained.

[0343] Fmoc-Glu-O t Bu-Lys(Boc)-O t Bu(7). To a mixture of Fmoc-Glu-OtBu(4) (4.26 g) and N-hydroxysuccinimide (1.15 g) in anhydrous THF (40 mL), DCC (2.06 g) was added all at once at room temperature. The resulting solution was stirred at room temperature overnight, then the solid was filtered off and washed with THF. The combined filtrate was evaporated to dryness and dried under vacuum to give crude Fmoc-Glu(OSu)-O t Bu(5) (5.3 g) as a white foam. This material was redissolved in THF (20 mL) and added at room temperature to a mixture of H-Lys(Boc)-OtBu-HCl(6) (3.39 g) and DIPEA (3.5 mL) in anhydrous THF (50 mL). The resulting mixture was stirred at ambient temperature for 4 h until the reaction was judged to be complete by HPLC analysis, and the solvent was removed under vacuum. The residue was redissolved in ethyl acetate (100 mL) and washed with 10% aqueous citric acid (50 mL), water (50 mL), and brine (50 mL), and dried over sodium sulfate. After removal of the solvent under vacuum, the residue was triturated with 5% ether / hexane (50 ml). The separated white solid product was filtered, washed with hexane, and dried under vacuum to give Fmoc-Glu-O t Bu-Lys(Boc)-O t Bu(7).

[0344] H-Glu-O t Bu-Lys(Boc)-O t Bu(8). The solution of H-Glu-O t Bu-Lys(Boc)-O t Bu(7) in DCM was treated with diethylamine. The resulting solution was stirred at room temperature for 4 h until deprotection was judged to be complete by HPLC analysis. All solvents were removed under vacuum and the residue was purified by silica gel column chromatography, eluting first with dichloromethane and then with 5 - 10% MeOH in dichloromethane to give H-Glu-O t Bu-Lys(Boc)-O t Bu(8) as a colorless oil.

[0345] Compound 9: A solution of amine 8 in anhydrous DMF was treated once with N 10 -trifluoroacetylpteroic acid OSu ester (3). The resulting mixture was stirred at room temperature for 24 h, during which completion was monitored by HPLC analysis. The reaction mixture was diluted with ethyl acetate and filtered through a pad of silica gel eluting with 10% methanol in ethyl acetate. The combined filtrates were evaporated to dryness, redissolved in ethyl acetate and washed successively with 10% aqueous citric acid, water, saturated NaHCO3 and brine. The extract was dried over sodium sulfate, evaporated and dried under vacuum overnight to give crude 9 as a brown solid.

[0346] Compound 10: Crude compound 9 was dissolved in a 1:1 (v / v) mixture of trifluoroacetic acid and dichloromethane. The resulting solution was left at room temperature for 2 h until complete deprotection was judged by HPLC analysis. All solvents were removed under vacuum and the residual brown oil was triturated with diethyl ether and sonicated briefly. The separated pale precipitate was collected by filtration, washed thoroughly with ether and dried under vacuum for 1 day to give product 10 as a pale yellow powder.

[0347] This example demonstrates the synthetic route for the synthesis of compound 13.

[0348]

Chemical formula

[0349] Compound 12: To a solution of compound 10 (0.45 g) and compound 11 (0.26 g) in DMF (15 mL) was added DIEA (0.44 mL) at room temperature. The reaction mixture was stirred at room temperature overnight until complete consumption of 11 was observed by HPLC analysis. The reaction mixture was diluted with pH 5 acetate buffer (0.5 M) and 1 mL of acetonitrile and purified by C18 reverse phase HPLC using a 20 - 90% acetonitrile / 0.05% TFA gradient as eluent, and compound 12 was obtained as a white solid after lyophilization.

[0350] Compound 13: Hydrazine, H2O (0.19 mL) was added to a solution of Compound 12 (0.31 g) in DMF (1.5 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (ca. 2 mL) and purified by C18 reverse-phase HPLC using a 20 - 90% acetonitrile / 0.05% TFA gradient as the eluent, and Compound 13 was obtained as a yellow solid after lyophilization. MS (ESI) m / z 831 (M + H) + 。

[0351] The present invention incorporates linker synthesis as exemplified below by demonstrating a synthetic route (CAS#: 1415328 - 95 - 8) for the synthesis of Compound 11.

[0352]

Chemical Structure

[0353] Compound 16: A small piece of sodium was added to a solution of tetraethylene glycol 14 in anhydrous THF at room temperature and stirred until completely dissolved. Acrylate 15 was slowly added to the resulting solution over 15 minutes. The reaction mixture was stirred at room temperature for 20 hours, then concentrated under vacuum, resuspended in brine, and subsequently extracted with ethyl acetate. The combined organic phases were washed with brine and dried over sodium sulfate. Removal of the solvent in vacuo gave Compound 16 as a clear yellowish oil.

[0354] Compound 17: p-Toluenesulfonyl chloride was added portionwise to a mixture of alcohol 16 and pyridine in anhydrous DCM at 0 °C. The resulting mixture was stirred at 0 °C for 30 minutes and then at room temperature overnight. The reaction mixture was quenched with 10% citric acid; the aqueous layer was extracted with ethyl acetate, and the combined organics were washed with saturated sodium bicarbonate, water, brine, and dried over sodium sulfate. After removal of the solvent, the residue was purified by silica gel to give tosylate 17 as a clear colorless oil.

[0355] Compound 18: To a mixture of tosylate 17 and N-hydroxyphthalimide in DMF, DBU was added at room temperature. The resulting dark red solution was heated to 90 °C for 1 hour, then cooled, quenched with 10% citric acid, and extracted with ethyl acetate. The organic phase was washed thoroughly with saturated aqueous sodium bicarbonate, water, and brine, and dried over sodium sulfate. After removing the solvent, the residue was purified by silica gel to give Compound 18 as a colorless oil.

[0356] Compound 19: The t-butyl ester 18 was treated with a 1:1 mixture of TFA and DCM at room temperature. After 3 hours, the solvent was removed under vacuum, the residue was taken up in dichloromethane, washed thoroughly with brine, and dried over sodium sulfate. After removing the solvent under vacuum, the crude carboxylic acid 19 was obtained as a pale yellowish transparent oil.

[0357] Compound 11: The crude carboxylic acid 19 in anhydrous THF was treated with N-hydroxysuccinimide, followed by treatment with DCC at room temperature. Stirring was continued for 6 hours, the solid was removed by filtration and washed with THF. The filtrate was evaporated, and the residue was passed through a silica pad and washed with EtOAc to give Compound 11 as a colorless oil, which gradually solidified to a white solid upon storage.

[0358] This example discloses the synthesis of a branched linker. For example, the synthetic route for the synthesis of Compound 25:

[0359]

Chemical formula

[0360] Compound 21: To a solution of OSu ester 11 (1.55 g) and Boc-Lys-OH 20 (0.72 g) in DCM (50 ml), DIEA (1.03 mL) was added at 23 °C. After 10 minutes, LCMS indicated that the reaction was complete. The mixture was washed with 1N HCl (50 ml), saturated sodium bicarbonate (50 ml), and brine (50 ml). The organic layer was dried over MgSO4. By removing the solvent, crude acid 21 was obtained as a white solid, which was used in the next step without purification.

[0361] Compound 22: The crude acid 21 was dissolved in anhydrous THF and treated with N-hydroxysuccinimide, followed by DCC at room temperature. The reaction mixture was stirred overnight at room temperature, then filtered to remove DCU and washed with THF. The product was isolated by silica gel column chromatography using a 0 - 10% methanol / DCM gradient as the eluent to give Compound 22 as a white solid (MS(ESI) m / z 737 (M+H) + ).

[0362] Compound 24: Compound 22 was dissolved in DCM and treated with Compound 23. The resulting mixture was treated with DIEA and stirred at room temperature for 5 hours. The reaction mixture was diluted with DCM, washed with water and brine, and dried over sodium sulfate. The crude product was purified with 5% citric acid (20 ml) and brine (50 ml). The organic layer was dried over MgSO4. The solvent was removed in vacuo to give Compound 24 as a white solid. The crude product was used in the next step without further purification. MS(ESI) m / z 887 (M+H) + .

[0363] Compound 25: Compound 24 was dissolved in THF and treated with N-hydroxysuccinimide, followed by DCC at room temperature. After 4 hours, the mixture was filtered to remove DCU and concentrated in vacuo. The residue was purified by silica gel column chromatography using a 0 - 6% methanol / DCM gradient as the eluent to give Compound 25 as a white solid (MS(ESI) m / z 984 (M+H) + ).

[0364] This example discloses a synthetic route for the synthesis of compound 30.

[0365]

Chemical formula

[0366] This example discloses the synthesis of branched PEG-folic acid compound 30.

[0367]

Chemical formula

[0368]

Chemical formula

[0369] Compound 26: To a solution of compound 25 (0.6 g, crude) and compound 10 (0.6 g) in DMF (5 ml), DIEA (0.47 mL) was added at 23 °C and stirred for 1 hour. The mixture was purified by Prep-LC using a 5% - 60% water / 90% ACN 0.05% TFA gradient over 20 minutes using a C18 column. The fractions containing the product were combined and evaporated in vacuo to give compound 26 as a brown solid; MS (ESI) m / z 1535 (M + H) + 。

[0370] Compound 27: To compound 26 (0.25 g), DCM (3 ml) and TFA (2 ml) were added at 23 °C, then stirred for 30 minutes. The solvent was removed in vacuo. The residue was dissolved in DCM (ca. 5 ml) and added dropwise to 45 ml of MTBE in a conical tube. The precipitate was isolated by centrifugation (4000 rpm, 5 minutes) and dried to give compound 27 as a brown solid; MS (ESI) m / z 1434 (M + H) + 。

[0371] Compound 29A: To a solution of Compound 27 (0.054 g) and Compound 28A (PEG5K-C5-NHS, 0.17 g) in DMF (2 ml), DIEA (0.034 mL) was added at 23 °C. After stirring for 5 hours, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 minutes, 4000 rpm) to separate the precipitate. 45 mL of MTBE was added to the precipitate and centrifuged (5 minutes, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to obtain Compound 29A as a crude white solid.

[0372] Compound 30A: To a solution of Compound 29A (0.24 g) in water (10 ml), hydrazine, H2O (0.033 ml) was added at 23 °C. After stirring for 24 hours, the mixture was purified by Prep-LC using a C18 column with a gradient of 20% - 100% ACN and 0.05% TFA in water for 20 minutes. The fractions containing the product were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to obtain Compound 30A as a pale yellow solid. (See, for example, Figure 12).

[0373] Compound 29B: To a solution of Compound 27 (0.04 g) and Compound 28B (PEG10K-C5-NHS, 0.26 g) in DMF (6 ml), DIEA (0.040 mL) was added at 23 °C. After stirring for 16 hours, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 minutes, 4000 rpm) to separate the precipitate. 45 mL of MTBE was added to the precipitate and then centrifuged (5 minutes, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to obtain Compound 29B as a crude white solid.

[0374] Compound 30B: To a solution of Compound 29B (0.47 g) in water (10 ml), hydrazine, H2O (0.080 ml) was added at 23 °C. After stirring for 6 hours, the mixture was purified by Prep-LC using a C18 column with a gradient of 20% - 100% ACN and 0.05% TFA in water for 20 minutes. The fractions containing the product were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to obtain Compound 30B as a pale yellow solid.

[0375] Compound 29C: To a solution of compound 27 (0.040 g) and compound 28C (PEG20K-C5-NHS, 0.51 g) in DMF (8 ml) was added DIEA (0.040 mL) at 23 °C. After stirring for 16 h, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 min, 4000 rpm) to separate the precipitate. 45 mL of MTBE was added to the precipitate and centrifuged again (5 min, 4000 rpm). The solvent was decanted and the white precipitate was dried under high vacuum overnight to obtain compound 29C as a crude white solid.

[0376] Compound 30C: To a solution of compound 29C (0.67 g, <0.031 mmol) in water (8 ml) was added hydrazine, H2O (0.060 ml) at 23 °C. After stirring for 6 h, the mixture was purified by Prep-LC using a C18 column with a gradient of 20% - 100% ACN and 0.05% TFA in water for 20 min. The fractions containing the product were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to obtain compound 30C as a pale yellow solid.

[0377] Compound 29D: To a solution of compound 27 (0.033 g, 0.023 mmol) and compound 28D ((PEG10K)2-C2-NHS, 0.4 g) in DMF (4 mL) was added DIEA (0.020 mL) at 23 °C. After stirring for 18 h, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 min, 4000 rpm) to separate the precipitate. 45 mL of MTBE was added to the precipitate and centrifuged (5 min, 4000 rpm). The solvent was decanted and the white precipitate was dried under high vacuum overnight to obtain compound 29D as a crude white solid.

[0378] Compound 30D: Hydrazine, H2O (0.080 mL) was added to a solution of Compound 29D (0.45 g, <0.021 mmol) in water (8 mL) at 23 °C. After stirring for 48 h, the mixture was purified by Prep-LC using a C18 column with a 20% - 100% ACN and 0.05% TFA in water gradient for 20 min. The fractions containing the product were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to give Compound 30D as a pale yellow solid.

[0379] Compound 28E: DIEA (0.010 mL) was added to a solution of Compound 28E1 ((PEG5K)2-NHS, 0.1 g) and aminovaleric acid (0.003 g) in DMF (0.5 mL) at 23 °C. After stirring for 1 h, the mixture was diluted to 1 mL with water and purified by a desalting column. The recovered fractions were lyophilized to give Compound 28E as a white solid.

[0380] Compound 30E: Compound 27 (0.009 g) was added to a solution of Compound 28E (0.04 g), DMTMMT (0.003 g) and DIEA (0.005 mL) in DMF (2 mL) at 23 °C. After stirring for 1 h, LCMS indicated that the reaction was complete. Hydrazine was added to this mixture (crude Compound 29E), and H2O (2 μL) was added in situ. After stirring for 1 h, the mixture was purified by Prep-LC using a C18 column with a 20% - 100% ACN and 0.05% TFA in water gradient for 20 min. The fractions containing the product were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to give Compound 30E as a pale yellow solid.

[0381] [Chemical Structure]

[0382] Compound 31: To a solution of 20K-PEG-amine (0.31 g) and 2,5-dioxopyrrolidin-1-yl 2-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)acetate (0.003 g) in DMF (1.0 ml), DIEA (0.006 mL) was added at 23 °C. After 30 minutes, the mixture was purified by a desalting column (PD-10) and lyophilized overnight to obtain Compound 31 as a white solid. Other PEG variants can be prepared using the same procedure described herein.

[0383] [Example 18] This example demonstrates the construction, expression, and purification of a dual amber-containing humanized anti-CD3 Fab lead molecule. A second pAF incorporation site was added to the anti-CD3 Fab light chain position LL157 of Fab1-HK129pAF, Fab9-HK129pAF, and Fab10-HK129pAF, resulting in new Fab molecules designated Fab1-HK129pAF-LL157pAF, Fab9-HK129pAF-LL157pAF, and Fab10-HK129pAF-LL157pAF, respectively. This enabled the complexation of two folic acids and two 5K PEG molecules in each Fab using any of the bifunctional linkers as described in the previous examples.

[0384] The CD3-PEG-folate purified proteins used in the in vitro activity, in vivo efficacy, and PK studies were analyzed by SDS gel electrophoresis (SDS PAGE). The purified CD3 was complexed with 5K or 10K PEG-folate at the pAF site using oxime chemistry and then purified. After complexation, cation exchange chromatography was utilized to separate the non-complexed, single-site, and two-site complexed forms (Figures 13A - 13B). After purification, the composition was formed in 50 mM histidine, 100 mM NaCl, 5% trehalose pH 6 and sterile filtered.

[0385] Figure 13A shows 5 μg of each purified CD3-folate bispecific antibody with 5K PEG conjugate. Lanes 3 and 6 represent non-conjugated CD3Fab compositions with single and double pAF incorporation, respectively. Lanes 4 and 7 represent CD3Fab compositions with single and double pAF and folate, respectively. Lanes 5 and 8 represent conjugated 5KPEG-folate and Bi5KPEG-Bi folate, respectively.

[0386] Figure 13B shows SDS-PAGE results under non-reducing conditions with 10 μg of protein per well. Lanes 2 and 7 show non-conjugated CD3Fab compositions, lanes 3 and 4 show different double-conjugated Bi5KPEG-Bi folate CD3Fab compositions, respectively, and lanes 5 and 6 show double-conjugated Bi10KPEG-Bi folate and 10KPEG-folate, respectively. The data (Figures 13A - B) show high purity (>90%) for all samples and the expected increase in molecular weight based on the size of PEG.

[0387] To enhance the conjugation efficiency, the purified CD3Fab composition was conjugated with PEG-folate using a two-step conjugation method. Folate was conjugated at the pAF site using oxime chemistry, followed by conjugating PEG with 5K, 10K, or 20K PEG at the C-terminal cysteines of both the heavy and light chains using maleimide-thiol click chemistry. Figure 13C shows SDS-PAGE under non-reducing (lanes 2 - 5) and reducing (lanes 7 - 10) conditions with 10 μg of protein per well. Lanes 2 and 7 represent Bi folate non-conjugated compositions, and lanes 3 and 8, 4 and 9, 5 and 10 represent Bi folate-C-terminal Bi5KPEG, Bi10KPEG, and Bi20KPEG compositions, respectively. The data in Figure 13C show high purity (>95%) for all samples and the expected increase in molecular weight based on the size of PEG.

[0388] Example 19 This example demonstrates the effect of Bi-folate and Bi-PEG conjugation in a CD3Fab1 composition and two low-affinity variants, Fab9 and Fab10. Human CD3 binding and cytotoxicity: Table 25 shows the effect of various modifications including Bi5KPEG-Bi folate conjugation on the binding affinity and cytotoxicity of Fab1 molecules in the presence of 50 nM SFA.

[0389]

Table 25

[0390] In vitro cytotoxicity assays were also performed in the presence of 50 nM SFA in KB, OV-90 and SKOV-3 cells using CD3-folate bispecific antibodies with 5K, 10K or 20K PEG C-terminal conjugates (Figure 13D, Table 26). Efficacy decreased slightly with increasing PEG length, but all constructs tested retained potent cytotoxic activity. Results and trends were consistent among all cell lines tested. Based on these studies, the slight decrease in efficacy observed with increasing PEG size is hypothesized to be offset by increased exposure from the extended half-life.

[0391]

Table 26

[0392] Table 27 compares the binding affinity of the parental Fab1 and two low-affinity variants, Fab9 and 10, to human CD3 in the presence of 20 nM SFA. As shown, conjugation of Bi5KPEG-Bi folate using a PEG-folate bifunctional linker resulted in a decrease in human CD3 binding and the corresponding potential for cytotoxicity for all three Fabs compared to their non-conjugated controls, as described in the above examples.

[0393]

Table 27

[0394] T cell activation and cytokine release: Table 27 shows the activation of the T cell marker CD69 and the release of two cytokines (IFNγ and TNFα) by three modified Fabs. As shown in the above examples, the general interrelationship between the strength of CD3 binding, the potential for cytotoxicity, T cell activation, and subsequent cytokine release is true across all Fabs, even after significant modification by bifolate-Bi5KPEG conjugation. This suggests that by reducing CD3 affinity while maintaining the effect and simultaneously increasing tumor-associated antigen (TAA) affinity, the toxicity due to cytokine release syndrome can be minimized. Furthermore, the PK properties, including half-life (T1 / 2) extension, were improved by site-specific PEGylation using a unique unnatural amino acid (UAA) incorporation technique.

[0395] Example 20 In vitro cytotoxicity data show that the CD3-folate bispecific antibody selectively kills FOLRα-expressing KB cells. KB cells were treated with increasing concentrations of the CD3-folate bispecific antibody in the presence of 50 nM folic acid (physiologically relevant concentration of folic acid). The most potent CD3-folate bispecific antibody, Fab1-HK129-LL157-Bifol ate, contains two molecules of folic acid and showed an IC 50 of 1.3 pM (Figure 14). The CD3-folate bispecific antibody containing a single folic acid, Fab1-HK129-folate, showed an IC50 of 40.3 pM and was not 31-fold more potent than Fab1-HK129-LL157-Bifol ate. This data shows that two folic acids increase the efficacy of the CD3-folate bispecific antibody. The addition of 5KPEG decreased the efficacy. A 4.9-fold decrease in efficacy was observed between the single CD3-folate bispecific antibody, Fab1-HK129-folate, and Fab1-HK129-5KPEG-folate, and the IC 50 value was 198 pM. A 37.3-fold decrease in efficacy was observed between Fab1-HK129-LL157-Bifol ate and Fab1-HK129-LL157-Bi5KPEG-Bifol ate, and the IC 50The value was 48.5 pM. These data indicated that the CD3-folate bispecific antibody retained potent in vitro cytotoxicity at physiologically relevant concentrations of folic acid.

[0396] [Example 21] In vitro cytotoxicity data indicate that the CD3-folate bispecific antibody selectively kills FOLRα-expressing SKOV3 cells in the presence of 20 or 50 nM folic acid. SKOV3 cells were treated with increasing concentrations of the CD3-folate bispecific antibody in 20 or 50 nM folic acid (physiologically relevant concentrations of folic acid) (Figures 15A and 15B). The CD3-folate bispecific antibody showed a 5.6- to 8-fold decrease in efficacy as the folic acid concentration increased from 20 nM to 50 nM, which is close to the maximum of the normal physiologically relevant concentrations of folic acid. The addition of 5KPEG also decreased the efficacy of the CD3-folate bispecific antibody. A 5.4-fold decrease was observed between the bispecific antibodies of single folic acid and single 5KPEG folic acid, and a 22.9-fold decrease was observed between the bispecific antibodies of double folic acid and double 5KPEG folic acid. No significant difference in efficacy was observed between the single and double pegylated CD3-folate bispecific antibodies. The data in Table 28 indicate that the CD3-folate bispecific antibody can kill FOLRα-expressing cells in the presence of physiologically relevant concentrations of folic acid, and that the pegylated antibody has a decreased efficacy compared to the non-pegylated antibody, but the CD3-folate bispecific antibody can kill FOLRα-expressing cells.

[0397] [Table 28]

[0398] [Example 22] Additional in vitro cytotoxicity assays were performed in the presence of 20 or 50 nM of 5-mTHF. SKOV3 cells were treated with increasing concentrations of the CD3-folate bispecific antibody in the presence of 20 or 50 nM (physiologically relevant concentrations of the major form of folate found in human serum), 5-methyltetrahydrofolate (5-mTHF) (Figures 16A and 16B). 5-mTHF has a binding affinity of 1 - 10 nM for FOLRα and does not bind to FOLRα, similar to folate which has a binding affinity of less than 1 nM. The CD3-folate bispecific antibody retained very potent IC50 values between 0.03 - 0.16 pM and 0.1 - 1.5 pM for the dual and single CD3-folate bispecific antibodies, respectively. The data in Table 29 show that the CD3-folate bispecific antibody has potent in vitro cytotoxicity against FOLRα-expressing SKOV3 cells in the presence of physiologically relevant concentrations of 5-mTHF.

[0399] [Table 29]

[0400] [Example 23] Mouse Pharmacokinetics Study in CD1 Mice: CD3-Folic Acid Bispecific Antibody was administered iv. at 1 or 5 mg / kg via the mouse tail vein of CD-1 mice. Blood samples were collected at 9 time points and analyzed by ELISA. The data clearly showed that the addition of 5KPEG increased serum exposure (AUClast) (Tables 30 - 31 and Figure 17). Fab1-HK129-5KPEG-Folic Acid showed a 4.3-fold improvement over Fab1-HK129-Folic Acid at 1 mg / kg, and Fab1-HK129-5KPEG-Folic Acid showed a 5-fold improvement over Fab1-HK129-Folic Acid at 5 mg / kg. Fab1-HK129-LL157-Bi5KPEG-Bi Folic Acid showed a 16.25-fold improvement over Fab1-HK129-LL157-Bi Folic Acid at 1 mg / kg, and Fab1-HK129-LL157-Bi5KPEG-Bi Folic Acid showed a 21.7-fold improvement over Fab1-HK129-LL157-Bi Folic Acid at 5 mg / kg. The data showed a 3.9-fold difference between Fab1-HK129-5KPEG-Folic Acid and Fab1-HK129-LL157-Bi5KPEG-Bi Folic Acid. When 5KPEG was added to the CD3-Folic Acid Bispecific Antibody, an improvement in serum half-life (T1 / 2) was also observed. The greatest improvement in serum half-life was observed with two 5KPEGs incorporated into the CD3-Folic Acid Bispecific Antibody. Fab1-HK129-LL157-Bi5KPEG-Bi Folic Acid showed a 4.2-fold improvement in serum half-life over Fab1-HK129-LL157-Bi Folic Acid at 1 mg / kg, and Fab1-HK129-LL157-Bi5KPEG-Bi Folic Acid showed a 6.25-fold improvement in serum half-life over Fab1-HK129-LL157-Bi Folic Acid at 5 mg / kg. The data indicate that the addition of 5KPEG improves serum exposure and the serum half-life of the CD3-Folic Acid Bispecific Antibody, reducing the dosing frequency to achieve effective serum exposure in vivo.

[0401]

Table 30

[0402]

Table 31

[0403] [[Example 24]] This example shows that the CD3-folate-folate bispecific antibody kills human M2 macrophages: Macrophages are a heterogeneous cell population that plays a role in host defense. Classically activated macrophages (M1 macrophages) have pro-inflammatory functions, recruitment of tumor-infiltrating lymphocytes, and anti-tumor activity, while M2 macrophages are anti-inflammatory and are involved in tissue remodeling, cancer cell migration, invasion, and metastasis. Human M2 macrophages are associated with cancer cell proliferation and poor prognosis in ovarian cancer. Inhibition of M2 macrophages can enhance the activity of immuno-oncology treatments such as checkpoint inhibitors. Human M2 macrophages express FOLRβ or FRβ, and these, or FR, have a binding affinity for folate similar to that of FOLRα or FRα. Therefore, strategies to increase the ratio of M1 macrophages to M2 macrophages by re-polarizing M1 macrophages or selectively killing M2 macrophages provide potential treatment approaches in cancer treatment.

[0404] Experiment 1: To evaluate the effect of the CD3-folate bispecific antibody on macrophages, the following experiment was performed: Fab1-HK129-LL157-pAF, Fab1-HK129-LL157-folate, and Fab1-HK129-LL157-5KPEG-folate were incubated with human M2 macrophages and human T cells in the presence of 50 nM folate. The data show that Fab1-HK129-LL157-folate and Fab1-HK129-LL157-5KPEG-folate had IC 50It shows killing of human M2 macrophages with the value (data not shown). Fab1-HK129-L157-pAF lacking folic acid does not kill human M2 macrophages. This data supports that the cytotoxicity of Fab1-HK129-LL157-folic acid and Fab1-HK129-LL157-5KPEG-folic acid is specific to binding to FOLRβ. Furthermore, the CD3-folic acid bispecific antibody suggests that in addition to killing FOLRα-expressing tumor cells, it can kill M2 macrophage cells and possibly other FOLRα / β-expressing immunosuppressive cells.

[0405] Test 2: In these tests, monocyte-derived macrophages were generated from human blood of healthy donors and treated with either granulocyte macrophage colony-stimulating factor (GM-CSF) for M1 macrophage differentiation or macrophage colony-stimulating factor (M-CSF) for M2 macrophage differentiation.

[0406] Before conducting the tests, folate receptor β (FOLRβ or FR-β) expression was first measured by flow cytometry and found to increase on average 26-fold in M2 macrophages compared to M1 macrophages with respect to the median fluorescence intensity (MFI).

[0407] Human M1 or M2 macrophages were seeded at 9,000 cells / well on 96-well clear-bottom white plates and incubated overnight. The next day, 90,000 human T cells were added as effector cells to the wells containing macrophages at an effector:target (E:T) cell ratio of 10:1, and incubated at 37 °C, 5% CO2 for 3 days in the presence of 20 nM folic acid with serial dilutions (0.001 pM to 100 nM) of the CD3-folic acid bispecific antibody. The relative viability of the macrophages was measured by CellTiter-Glo (100 μL / well) after removal of floating cells and calculated as a percentage of the untreated control.

[0408] Effect of PEGylation of CD3-folate bispecific antibody: Table 32 shows the IC 50 data for in vitro macrophage cytotoxicity by CD3-folate bispecific antibody containing single folate in the presence of 20 nm folate. Fab1-HK129-folate had an average IC 50 of 85.0 pM (range 53.5 - 120.0 pM) in M1 macrophages and an average IC 50 of 5.6 pM (range 1.4 pM - 15.0 pM) in M2 macrophages. Based on the IC 50 ratio, M2 macrophages were on average 26-fold improved (range 5 - 42-fold) with Fab1-HK129-folate compared to M1. Fab1-HK129-5KPEG-folate had an average IC 50 of 616.8 pM (range 198.0 - 908.9 pM) on average in M1 macrophages and an average IC 50 of 13.4 pM (range 2.3 - 33.2 pM) on average in M2 macrophages, showing an improved average IC 50 ratio between M1 and M2 macrophages, and an average 69-fold (range 23 - 126-fold) improvement in M2 macrophages. These results indicate that CD3-folate bispecific antibody containing single folate is specific for killing M2 macrophages, and PEGylation of CD3-folate bispecific antibody containing single folate (Fab1-HK129-5KPEG-folate) is more selective for killing M2 macrophages than Fab1-HK129-folate.

[0409]

Table 32

[0410] Results of in vitro macrophage cytotoxicity by CD3-folate bispecific antibody containing single folate from donor 6007, a representative donor, are shown in Figure 18A. Arrows and numbers represent the difference (magnification) between M1 and M2 macrophages in terms of IC 50 The dotted line indicates Fab1-HK129-folate in M1 and M2, and the solid line indicates Fab1-HK129-5KPEG-folate in M1 and M2.

[0411] Table 33 shows the IC data for in vitro macrophage cytotoxicity by the CD3-folate bispecific antibody containing dual folate in the presence of 20 nM folic acid. 50 Fab1-HK129-LL157-Bi folate has an average IC of 29.2 pM (range 10.9 - 42.8 pM) in M1 macrophages and an average IC of 1.5 pM (range 0.1 pM - 5.5 pM) in M2 macrophages, and the ratio of the average IC between M1 and M2 macrophages is 72-fold different (range 6 - 212-fold). 50 (range 10.9~42.8pM), with an average IC of 1.5 pM in M2 macrophages 50 (range 0.1 pM - 5.5 pM), and the average IC between M1 and M2 macrophages 50 The ratio is 72-fold different (range 6 - 212-fold). Fab1-HK129-LL157-Bi folate-Bi5KPEG has an average IC of 1620.8 pM (range 834.7 - 2651 pM) in M1 macrophages and an average IC of 15.7 pM (range 3.6 - 52.4 pM) in M2 macrophages, and the average IC between M1 and M2 macrophages 50 (range 834.7~2651pM), with an average IC of 15.7 pM in M2 macrophages 50 (range 3.6 - 52.4 pM), and the average IC between M1 and M2 macrophages 50 The ratio is improved and is 181-fold (range 49 - 501-fold) in M2 macrophages. These results indicate that the CD3-folate bispecific antibody containing dual folate is specific for killing M2 macrophages. PEGylation of the CD3-folate bispecific antibody containing dual folate (Fab1-HK129-LL157-Bi folate-Bi5KPEG) is shown to be less effective than Fab1-HK129-LL157-Bi folate or Fab1-HK129-5KPEG-folate as shown in Table XXX, but is shown to be more selective for killing M2 macrophages.

[0412]

Table 33

[0413] Figure 18B shows the results of in vitro macrophage cytotoxicity by the CD3-folate bispecific antibody containing dual folate from donor 6007, a representative donor. The arrows and numbers indicate the difference (magnification) between M1 and M2 macrophages at the IC 50It is represented by. The dotted line indicates Fab1-HK129-LL157-Bi folic acid in M1 and M2, and the solid line indicates Fab1-HK129-LL157-Bi folic acid-Bi5KPEG in M1 and M2.

[0414] To mimic the physiologically relevant concentrations of the main forms of folic acid found in human serum in the range of 9.1 - 45.1 nM, further studies were conducted. Of this physiological range, 86.7% (37.5 nM) is 5-methyl-tetrahydrofolic acid (5-mTHF), the primary folic acid metabolite, and only 4% (1.2 nM) is unmetabolized folic acid (Pfeiffer et al., Br. J. Nutr., 2015 June 28:113(12):1965 - 1977). As is well known in the art, the binding affinity of folic acid for FR-β is less than 1 nM, and the affinity of 5-mTHF for FR-β is 1 - 10 nM. From this, it was hypothesized that the concentration or composition of folic acid and 5-mTHF could affect the activity of the CD3-folic acid bispecific antibody. To evaluate this, the following experiment was conducted: In vitro macrophage cytotoxicity data in the presence of 45 nM of 5-mTHF are shown in Tables 34 and 35. The average IC 50 for Fab1-HK129-folic acid was 9.1 pM and 0.31 pM in M1 macrophages and M2 macrophages, respectively, and the average IC 50 for Fab1-HK129-5KPEG-folic acid was 48.5 pM and 1.26 pM in M1 macrophages and M2 macrophages, respectively. The average IC 50 for Fab1-HK129-LL157-Bi folic acid was 4.0 pM in M1 and 0.05 pM in M2, and the average IC 50 for Fab1-HK129-LL157-Bi folic acid-Bi5KPEG was 99.4 pM in M1 and 0.85 pM in M2. These data indicate that the CD3-folic acid bispecific antibody is more potent in the presence of 5-mTHF than folic acid (Tables 32 and 33). The IC 50The ratios were an average of 43-fold and 57-fold for the CD3-folate bispecific antibody containing single folic acid, and an average of 89-fold and 212-fold for the CD3-folate bispecific antibody containing dual folic acid. These results indicate that CD3-folate containing bispecific antibodies maintain M2 macrophage specific killing, and PEGylation confers more specificity at physiologically relevant concentrations of 5-mTHF.

[0415]

Table 34

[0416]

Table 35

[0417] Overall, the data indicate that in the presence of folic acid or 5mTHF metabolites, the dual PEGylated CD3-folate composition showed greater specificity for the killing of M2 macrophages than the single PEGylated CD3-folate composition. Thus, improved selectivity was observed with more PEGylation.

[0418] Further studies with CD3-folate bispecific antibodies were also performed on human myeloid-derived suppressor cells (MDSC) to examine FRβ expression. Human peripheral blood mononuclear cells (PBMC) from healthy donors were treated with Fab1-HK129-5KPEG-folate and Fab1-LL157-Bi5KPEG-Bi folate, and the cytotoxic activity against mononuclear MDSC (mMDSC) at 0, 1, 10, and 100 pM was tested for each CD3-folate bispecific antibody. The cells were added to PBMC and incubated in a 37 °C and 5% CO2 incubator for 24 hours. After incubation, mMDSC were gated by the CD3 - / CD33 + / CD11b + / CD14 + / HLA-DR low population using flow cytometry, and the percentage of live cells from untreated controls was measured.

[0419] Based on the observations, the proportion of mMDSC decreased in a dose-dependent manner. Fab1-HK129-5KPEG-folic acid showed a dose-dependent decrease from 72% to 24% at 10 pM to 100 pM, respectively. Similarly, Fab1-HK129-LL157-Bi5KPEG-Bi folic acid showed a dose-dependent decrease from 88% to 22% at 10 pM to 100 pM, respectively. Considering that non-MDSC with high HLA-DR were preserved, treatment with the CD3-folic acid bispecific antibody was shown to selectively eliminate ...

Claims

[Claim 1] The invention described in the specification.

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