Anti-CD3 antibody folate bioconjugates and uses thereof

Abstract

Novel anti-CD3 folate antibodies and their use in the treatment of diseases or conditions that may benefit from such antibodies are described.

Description

Detailed Description of the Invention

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 732,793, filed August 28, 2018, entitled "Anti-CD3 Fab Folates Antibodies and Their Uses," the contents of which are incorporated herein by reference in their entirety.

[0002] [Sequence Listing] This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on August 28, 2019 is titled AMBX_0228_00PCT_Sequence_Listing.txt and is 128,417 bytes in size.

[0003] FIELD OF THE INVENTION The present disclosure relates to the field of immuno-oncology. More specifically, the present invention relates to anti-CD3 antibodies and fragments or variants thereof conjugated to one or more folate molecules. The present invention also relates to anti-CD3 Fab-folate antibodies and variants thereof conjugated to 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 annually, and approximately 140,000 women die from the disease each year. Five-year survival rates for ovarian cancer vary by cancer type and stage, with a trend toward poorer five-year survival rates for more advanced ovarian cancer. Current treatment options for ovarian cancer include chemotherapy, surgery, radiation, or a combination of these therapies. Following debulking surgery followed by platinum-based chemotherapy, the response rate for advanced ovarian cancer is 80%, with 40–60% achieving complete responses. Unfortunately, approximately 70% of these patients experience recurrence, and the median progression-free survival is 18 months.

[0005] Currently, there are shortcomings in the field of targeted therapies for ovarian cancer, and types of ovarian cancer including eptithelial, stromal, and germ cell tumors, as well as fallopian tube and primary peritoneal carcinoma. While surgery, radiation, and various chemotherapy drugs are commonly used to treat patients with ovarian cancer, the FDA (U.S. Food and Drug Administration) recently accepted the supplemental Biologics License Application (sBLA) for bevacizumab (Avastin) for the treatment of advanced ovarian cancer as a first-line therapy.

[0006] Immunotherapy is being evaluated as a new treatment option for cancer patients, using biologics or engineered T cells to stimulate the patient's immune system to combat cancer. Several immunotherapies have shown great promise 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 (BiTEs), various designs of T cell-dependent bispecific antibodies (TDBs), NK cell-dependent bispecific antibodies, macrophage-dependent bispecific antibodies, and autologous antigen-presenting cell (APC) / cancer vaccines.

[0007] Biologics currently undergoing clinical trials in patients with ovarian cancer include an antibody-drug conjugate against folate receptor alpha (mirvetuximab soravtansine (IMGN853)), an EpCAM-CD3 bispecific antibody (catumaxomab), a DLL4-VEGF bispecific vaccine targeting NY-ESO-1, and a CAR-T targeting folate receptor alpha (FOLR1).

[0008] Ovarian cancer has been reported to have an immunosuppressive environment, which has made the development of certain types of immunotherapeutic agents, such as checkpoint inhibitors, a challenge. Currently, no immunotherapeutic agents have been approved for treating patients with ovarian cancer, although several are being evaluated in clinical trials.

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

[0010] To overcome the shortcomings in the art, the present inventors have developed antibody conjugates in which an anti-CD3 antibody incorporates one or more non-naturally encoded amino acids and further comprises one or more folate molecules. Bispecific antibodies can comprise or consist of anti-CD3 antibodies engineered to contain one or more non-naturally encoded amino acids in the heavy or light chain of the Fab. Bispecific antibodies or antibody fragments can comprise anti-CD3 antibodies engineered to contain 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 recruit cytotoxic T cells to cancer cells are a promising new approach for 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 a non-naturally encoded amino acid in the heavy or light chain of the Fab, preferably in the heavy chain. In some embodiments, the non-naturally encoded amino acid is paraacetylphenylalanine (pAF). In some embodiments, the anti-CD3 antibody comprises two or more non-naturally encoded amino acids in the heavy and / or light chain of the Fab, e.g., two, three, or four non-naturally encoded amino acids, optionally, the two or more non-naturally encoded amino acids are pAF. In some embodiments, the anti-CD3 Fab is conjugated to a folate. In some embodiments, the anti-CD3 Fab is conjugated to a water-soluble polymer, such as polyethylene glycol (PEG). In some embodiments, the anti-CD3 Fab is conjugated to both folate and polyethylene glycol (PEG), optionally conjugated using a bifunctional linker. In some embodiments, the anti-CD3 Fab is conjugated to two folate and two polyethylene glycol (PEG) molecules. In some embodiments, conjugation is via the side chain of a non-naturally encoded amino acid (e.g., pAF). In some embodiments, the anti-CD3 antibody recruits cytotoxic T cells to folate receptor-positive (FR+) tumor cells. In some embodiments, the anti-CD3 antibody has improved potency, 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 potency. 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 activity. 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 optimized anti-CD3 Fab-folate conjugates that target cytotoxic T cells to folate receptor-positive (FR+) tumor cells, resulting in optimal efficacy, reduced toxicity, and optimal pharmacokinetic (PK) properties. For example, the present invention provides optimized anti-CD3 Fab-folate conjugates that target cytotoxic T cells to folate receptor-positive (FR+) tumor cells, resulting in optimal efficacy, reduced toxicity, and optimal pharmacokinetic (PK) properties. To achieve an optimal balance between efficacy and toxicity due to cytokine release syndrome (CRS), we 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), we simultaneously conjugated both folate and PEG molecules of various sizes using a bifunctional linker. The optimized conjugates demonstrated potent and selective in vitro activity, good serum half-life, and robust in vivo activity in xenograft mouse models. This semisynthetic 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 anti-CD3 antibodies and conjugates of the anti-CD3 antibodies with folate. The present invention also provides conjugates of the anti-CD3 antibodies with PEG. The present invention also provides conjugates of the anti-CD3 antibodies with folate and PEG. In some embodiments, the novel anti-CD3 antibodies of the present invention comprise one or more non-naturally encoded amino acids. In some embodiments, the anti-CD3 antibodies comprise a complete antibody heavy chain. In some embodiments, the anti-CD3 antibodies comprise a complete antibody light chain. In some embodiments, the anti-CD3 antibodies comprise a complete antibody heavy chain and a complete antibody light chain. In some embodiments, the anti-CD3 antibodies comprise a variable region of an antibody light chain. In some embodiments, the anti-CD3 antibodies comprise a variable region of an antibody heavy chain. In some embodiments, the anti-CD3 antibodies comprise a variable region of a light chain and a variable region of a heavy chain. In some embodiments, the anti-CD3 antibodies comprise one or more CDRs of an antibody light chain. In some embodiments, the anti-CD3 antibodies comprise one or more CDRs of an anti-CD3 antibody heavy chain. In some embodiments, the anti-CD3 antibody comprises one or more CDRs of the light chain and one or more CDRs of the heavy chain. In some embodiments, the anti-CD3 antibody comprises three CDRs of the light chain. In some embodiments, the anti-CD3 antibody comprises three CDRs of the heavy chain. In some embodiments, the anti-CD3 antibody comprises three CDRs of the light chain and three CDRs of the heavy chain. In some embodiments, the anti-CD3 antibody comprises a Fab. In some embodiments, the anti-CD3 antibody comprises two Fabs. In some embodiments, the anti-CD3 antibody comprises two or more Fabs. In some embodiments, the anti-CD3 antibody comprises an scFv. In some embodiments, the anti-CD3 antibody comprises two scFvs. In some embodiments, the anti-CD3 antibody comprises two or more scFvs. In some embodiments, the anti-CD3 antibody comprises a minibody. In some embodiments, the anti-CD3 antibody comprises two minibodies. In some embodiments, the anti-CD3 antibody comprises two or more minibodies. In some embodiments, the anti-CD3 antibody comprises 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 light chain variable region and a heavy chain variable region. 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 embodiments. In some embodiments, the anti-CD3 antibody comprises a homodimer, heterodimer, homomultimer, or heteromultimer of any of the above-described embodiments. In some embodiments, the anti-CD3 antibody comprises a polypeptide conjugated to a binding partner (wherein the binding partner comprises an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance). In some embodiments, the anti-CD3 antibody is conjugated to a scaffold molecule or substance other than an antibody.

[0013] In some embodiments, the anti-CD3 antibody comprises one or more post-translational modifications. In some embodiments, the anti-CD3 antibody is linked to a linker, polymer, or biologically active molecule. In some embodiments, the anti-CD3 antibody is linked to a bifunctional polymer, 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, the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides (which may also comprise a non-naturally encoded amino acid). In some embodiments, the antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more polypeptide-small molecule conjugates (which may also comprise a non-naturally encoded amino acid). In some embodiments, the anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides (which may also comprise a non-naturally encoded amino acid).

[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 more than one small molecule ligand. In some embodiments, the small molecule ligand comprises a folate molecule or a DUPA molecule. In some embodiments, the small molecule ligand comprises two folate molecules or two DUPA molecules. In some embodiments, the small molecule ligand comprises two or more folate 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 comprises 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, amino acid substitutions in an anti-CD3 antibody can be with naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is with a non-naturally encoded amino acid.

[0016] In some embodiments, the non-naturally encoded amino acid comprises 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) molecules is about 0.1 kDa to about 100 kDa. In some embodiments, the average molecular weight of the poly(ethylene glycol) molecules is 0.1 kDa to 50 kDa. In some embodiments, the average molecular weight of the poly(ethylene glycol) is 1 kDa to 25 kDa, 2 to 22 kDa, or 5 kDa to 20 kDa. For example, the average molecular weight can be about 5 kDa, about 10 kDa, or about 20 kDa. For example, the average molecular weight of the poly(ethylene glycol) polymer can be 5 kDa, 10 kDa, or 20 kDa. In certain embodiments, the molecular weight is measured by an appropriate 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 1 kDa to 100 kDa, or 1 kDa to 50 kDa. In some embodiments, the molecular weight of each branch of the poly(ethylene glycol) branched polymer is 1 kDa to 25 kDa, 2 to 22 kDa, or 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, 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, 10 kDa, or 20 kDa.

[0019] The invention also provides anti-CD3 antibody polypeptides comprising a linker, polymer, or biologically active molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acids are incorporated into the polypeptide by the ribosome at a preselected position.

[0020] An embodiment of the present invention provides an anti-CD3 antibody having one or more of SEQ ID NOs: 1 to 62. An anti-CD3 antibody having two of SEQ ID NOs: 1 to 62. An embodiment of the present invention provides 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. An embodiment of the present invention provides 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 invention provides a bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain 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, and any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. Another embodiment of the invention provides a bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain 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.Another embodiment of the invention provides a bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain 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. Another embodiment of the invention provides a cytotoxically active CD3-specific binding construct having the amino acid sequence set forth in one or more of SEQ ID NOs: 1 to 62. Another embodiment of the invention provides a cytotoxically active CD3-specific binding construct having the amino acid sequence set forth in two of SEQ ID NOs: 1 to 62. Another embodiment of the invention provides a cytotoxically 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. Another embodiment of the present invention provides a cytotoxically active CD3Fab specific binding construct comprising an anti-CD3Fab 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 cytotoxically active CD3-specific binding constructs 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. Another embodiment of the present invention provides an anti-CD3 Fab antibody, the anti-CD3 antibody having 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] Another embodiment of the present invention provides an anti-CD3 antibody, the antibody comprising one or more post-translational modifications. Another embodiment of the present invention provides an anti-CD3 Fab antibody, the antibody being linked to a linker, polymer, or biologically active molecule. Another embodiment of the present invention provides an anti-CD3 antibody, the biologically active molecule being a folate. Another embodiment of the present invention provides an anti-CD3 antibody comprising one or more folates. Another embodiment of the present invention provides an anti-CD3 antibody comprising two folates. Another embodiment of the present invention provides an anti-CD3 antibody, the biologically active molecule being a DUPA. Another embodiment of the present invention provides an anti-CD3 antibody comprising one or more DUPAs. Another embodiment of the present invention provides an anti-CD3 antibody comprising two DUPAs.

[0022] In certain embodiments, the anti-CD3 antibody has a heavy chain amino acid sequence of 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 a light chain amino acid sequence of any one 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 invention provide anti-CD3 variants 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 NOs: 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 NOs: 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 comprises one or more unnatural amino acids. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NOs: 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 comprises two unnatural amino acids.In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 7, which comprises one or more unnatural amino acids; and one of SEQ ID NOs: 10, 14, 11, 15, 12, 16, 13, 17, 1, 18, or 19, which comprises one or more unnatural amino acids. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NO: 12, which comprises one or more unnatural amino acids; and one of SEQ ID NOs: 18, 19, or 16, which comprise one or more unnatural amino acids. In some embodiments, the anti-CD3 antibody comprises an anti-CD3 Fab having SEQ ID NOs: 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 unnatural amino acids, wherein the antibody has SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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 NOs: 6 and 7, or 6 and 8, or 6 and 9. The anti-CD3 antibody can be a bispecific antibody having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain comprises the anti-CD3 Fab. Another embodiment of the invention provides an anti-CD3 Fab antibody selected from SEQ ID NOs: 1-62, wherein a non-naturally encoded amino acid has been incorporated into the antibody. In some embodiments, the non-naturally encoded amino acid has been site-specifically incorporated into the antibody.

[0026] Other embodiments provide anti-CD3 Fab antibodies, wherein 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-GlcNAc β-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 comprising site-specifically incorporated non-naturally encoded amino acids.

[0014] Embodiments of the present invention provide anti-CD3 Fab antibodies comprising site-specifically incorporated non-naturally encoded amino acids at any one of positions H114, H115, H129, L157, H160, L172, and L205 (according to Kabat numbering, which is well known to those of skill in the art).

[0015] Embodiments of the present invention provide anti-CD3 Fab antibodies comprising site-specifically incorporated non-naturally encoded amino acids at positions H114, H115, H129, L157, H160, L172, and L205 (according to Kabat numbering, which is well known to those of skill in the art).

[0016] Embodiments of the present invention provide anti-CD3 Fab antibodies comprising site-specifically incorporated non-naturally encoded amino acids at position 114.

[0017] Embodiments of the present invention provide anti-CD3 Fab antibodies comprising site-specifically incorporated non-naturally encoded amino acids at position 115. Embodiments of the invention provide anti-CD3 Fab antibodies comprising a site-specifically incorporated non-naturally encoded amino acid at position 129. Embodiments of the invention provide anti-CD3 Fab antibodies comprising a site-specifically incorporated non-naturally encoded amino acid at position 157. Embodiments of the invention provide anti-CD3 Fab antibodies comprising a site-specifically incorporated non-naturally encoded amino acid at position 160. Embodiments of the invention provide anti-CD3 Fab antibodies comprising a site-specifically incorporated non-naturally encoded amino acid at position 172. Embodiments of the invention provide anti-CD3 Fab antibodies comprising a site-specifically incorporated non-naturally encoded amino acid at position 205. Other embodiments of the invention provide anti-CD3 Fab antibodies comprising one or more non-naturally encoded amino acids. Other embodiments of the invention provide anti-CD3 Fab antibodies comprising two non-naturally encoded amino acids. Other embodiments of the 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 contain one non-naturally encoded amino acid in the light chain, other embodiments of the invention provide anti-CD3 Fab antibodies that contain one non-naturally encoded amino acid in the heavy chain, and other embodiments of the invention provide anti-CD3 Fab variants that contain two non-naturally encoded amino acids.

[0028] Another embodiment of the invention provides an anti-CD3 Fab variant wherein the heavy chain further comprises an amino acid extension at the C-terminus. Another embodiment of the invention provides an anti-CD3 Fab variant wherein the amino acid extension comprises the amino acids DKTHT. Another embodiment of the invention provides an anti-CD3 Fab variant wherein the heavy chain further comprises an amino acid extension at the C-terminus. Another embodiment of the invention provides an anti-CD3 Fab variant wherein the amino acid extension comprises the amino acids DKTHT.

[0029] Other embodiments of the invention provide anti-CD3 Fab antibodies whose heavy chain sequences have murine framework residues at one or more positions for antigen binding. Other embodiments of the invention provide anti-CD3 Fab antibodies whose heavy chain sequences have murine framework residues at positions 30, 49, 77, or 93 (according to Kabat numbering). Other embodiments of the invention provide anti-CD3 Fab antibodies whose light chain sequences have murine framework residues at one or more positions. Other embodiments of the invention provide anti-CD3 Fab antibodies whose light chain sequences have framework residues at positions 36, 46, 49, 57, or 58 (according to 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 anti-CD3 Fab antibodies wherein the linker is a water-soluble polymer. Other embodiments of the present invention provide anti-CD3 Fab antibodies wherein the water-soluble polymer comprises poly(ethylene glycol). Other embodiments of the present invention provide anti-CD3 Fab antibodies wherein the water-soluble polymer is a linear or branched water-soluble polymer. Other embodiments of the present invention provide anti-CD3 Fab antibodies wherein the water-soluble polymer is linked to a non-naturally encoded amino acid present in the antibody. In some embodiments, the linker is attached to the anti-CD3 Fab antibody via the side chain of the non-natural amino acid. Other embodiments of the present invention provide anti-CD3 Fab antibodies comprising two or more amino acids linked to a water-soluble polymer comprising poly(ethylene glycol). Other embodiments of the present invention provide anti-CD3 Fab antibodies wherein one or more amino acids linked to the water-soluble polymer are non-naturally encoded amino acids. Other embodiments of the present invention provide anti-CD3 Fab antibodies comprising one or more poly(ethylene glycol). Other embodiments of the present invention provide anti-CD3 Fab antibodies wherein the poly(ethylene glycol) is between 1 kDa and 100 kDa. Another embodiment of the invention provides an anti-CD3 Fab antibody comprising one or more folates and one or more poly(ethylene glycols).Another embodiment of the invention provides an anti-CD3 Fab antibody comprising two folates and two poly(ethylene glycols).

[0031] Another embodiment of the present invention provides a method for optimizing cell killing in cells that overexpress folate receptors, the method comprising an anti-CD3 Fab antibody, the antibody comprising one or more folates, and one or more non-naturally encoded amino acids incorporated into the antibody. Another embodiment of the present invention provides a method for optimizing killing of tumor cells that overexpress folate receptors by redirected T cells, the method comprising an anti-CD3 Fab antibody, the antibody comprising one or more folates, and one or more non-naturally encoded amino acids incorporated into the antibody. In another embodiment, the method further comprises one or more water-soluble polymers. Another embodiment of the present invention provides a method wherein the water-soluble polymer comprises poly(ethylene glycol). In another embodiment, the water-soluble polymer is a linear or branched water-soluble polymer. The poly(ethylene glycol) is between 1 kDa and 100 kDa. The poly(ethylene glycol) is 5, 10, 20, 30, 40, 50, or 60 kDa. In another embodiment, the number of folate receptors is 10,000 or more.

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

[0033] Other embodiments of the invention provide anti-CD3 Fab antibodies in which the heavy or 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 (according to 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 (according to Kabat numbering).

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

[0035] Another embodiment provides a method of treating a patient having a disease or condition in cells that overexpress the folate receptor, comprising administering to the patient a therapeutically effective amount of an anti-CD3 Fab antibody described herein. Another embodiment of the present invention provides a bispecific anti-CD3 Fab comprising two folate molecules and two PEGylated molecules. In another embodiment, the bispecific anti-CD3 Fab antibody further comprises a site-specifically incorporated non-naturally encoded amino acid. The present invention provides a method of treating cancer by administering to the patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is an eptithelial tumor, a stromal tumor, or 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 alpha (FOLR1), such as ovarian cancer. In some embodiments, the cancer is treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The present invention provides a method for treating a genetic disease by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The present invention provides a method for treating AIDS by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The present invention provides a method for treating diabetes by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The anti-CD3 antibody of the present invention can be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally conjugated to two folate molecules and two PEGylated molecules. In a further embodiment, a non-naturally encoded amino acid is site-specifically incorporated into the anti-CD3 Fab antibody, and the anti-CD3 Fab antibody is conjugated to two folate molecules and two PEGylated molecules via the side chain of the non-natural amino acid.

[0036] The anti-CD3 antibodies of the present invention are used to treat diseases or conditions in cells that highly express folate receptors. The anti-CD3 antibodies of the present invention are used to treat cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is an eptithelial tumor, a stromal tumor, or 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 alpha (FOLR1), such as ovarian cancer. In some embodiments, the cancer is treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The anti-CD3 antibodies of the present invention are used to treat genetic diseases. The anti-CD3 antibodies of the present invention are used to treat AIDS. The anti-CD3 antibodies of the present invention are used to treat diabetes. The anti-CD3 antibodies of the present invention may be bispecific antibodies comprising an anti-CD3 Fab antibody, which may optionally be conjugated to two folate molecules and two PEGylated molecules. In further embodiments, a non-naturally encoded amino acid is site-specifically incorporated into the anti-CD3 Fab antibody, and the antibody is conjugated to two folate molecules and two PEGylated molecules via the side chain of the non-natural amino acid. The anti-CD3 antibodies of the invention can be used in the manufacture of a medicament for treating a disease or condition in cells that highly express folate receptors. The anti-CD3 antibodies of the 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 eptithelial tumor, a stromal tumor, or 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 alpha (FOLR1), such as ovarian cancer. In some embodiments, the cancer is treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The anti-CD3 antibodies of the invention can be used in the manufacture of a medicament for treating a genetic disease. The anti-CD3 antibodies of the invention can be used in the manufacture of a medicament for treating AIDS. The anti-CD3 antibodies of the invention can be used in the manufacture of a medicament for treating diabetes.The anti-CD3 antibodies of the invention can be bispecific antibodies comprising an anti-CD3 Fab antibody, optionally conjugated to two folate molecules and two PEGylated molecules. In a further embodiment, a non-naturally encoded amino acid has been site-specifically incorporated into the anti-CD3 Fab antibody, and the anti-CD3 Fab antibody is conjugated to two folate molecules and two PEGylated molecules via the side chains of the non-natural amino acid.

[0037] The present disclosure provides anti-CD3 antibodies, antibody fragments, or variants 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), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules with other scaffolds, bispecific antibodies, etc. In some embodiments, the anti-CD3 antibody, antibody fragment, or variant is an anti-CD3 Fab antibody, fragment, or variant.

[0038] The present disclosure provides a bispecific antibody conjugate comprising an anti-CD3 antibody or antibody fragment. The present invention also discloses a bispecific antibody conjugate comprising an anti-CD3 Fab antibody or antibody fragment incorporating one or more non-naturally encoded amino acids. Furthermore, the present invention describes a bispecific antibody conjugate comprising an anti-CD3 Fab antibody or antibody fragment and one or more folate molecules, wherein one or more non-naturally encoded amino acids are site-specifically incorporated into the anti-CD3 Fab antibody. The present disclosure also describes a bispecific antibody conjugate comprising an anti-CD3 Fab antibody or antibody fragment, one or more folate 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. The present disclosure also describes 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 one or more linkers, and the small molecules are one or more folate molecules or one or more DUPA molecules, or analogs or derivatives. The antibody or antibody fragment may be site-specifically linked to one or more folate molecules or one or more DUPA molecules by one or more linkers. The antibody or antibody fragment may comprise one or more unnatural amino acids. The antibody or antibody fragment may be site-specifically linked to one or more folate molecules or one or more DUPA molecules by one or more linkers to one or more unnatural amino acids. The unnatural amino acids may be site-specifically incorporated into the antibody. The antibody or antibody fragment may be site-specifically linked to one or more folate molecules or one or more DUPA molecules by one or more PEG molecules to one or more unnatural amino acids. Alternatively, the antibody or antibody fragment may be site-specifically linked to one or more folate molecules or one or more DUPA molecules by one or more linkers to natural amino acids. The antibody or antibody fragment may be an anti-CD3 Fab.

[0039]

[0010] A bispecific antibody conjugate is described that includes an anti-CD3 Fab, one or more folate molecules, and one or more linkers, wherein the anti-CD3 Fab is linked to the one or more folate molecules by one or more linkers. The anti-CD3 Fab can include one or more unnatural amino acids. The one or more unnatural amino acids can replace natural amino acids in the anti-CD3 Fab.

[0011] A bispecific antibody conjugate is described that includes 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 one or more linkers. The anti-CD3 Fab can include one or more unnatural amino acids. The one or more unnatural amino acids can replace natural amino acids in the anti-CD3 Fab.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0041] Figure 1 shows humanized anti-CD3 Fab binding to human PBMC. The figure shows the binding of humanized anti-CD3 Fab expressed in HEK293 cells to human PBMC (two experiments). A total of nine Fabs were tested by combining three vH chain sequences and three vL chain sequences. Three Fabs with the vL1.0 chain lost binding to human CD3 in all combinations with the vH chain.

[0042] Figures 2A-2F show titrations of humanized anti-CD3 Fab binding to human and canine PBMCs: (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 pharmacokinetic (PK) analysis of anti-CD3 Fab1 molecules in rats. PEGylation of humanized anti-CD3 Fab1 molecules extends the plasma half-life (T1 / 2) in rats.

[0044] Figures 4A-4B show the binding of first-round low-affinity Fabs derived from HEK293 cells. First-round anti-CD3 Fab mutants generated from HEK293 cells with low binding affinity to human CD3 (Figure 4A) and canine CD3 (Figure 4B) are shown. Titration curves for four selected low-affinity Fabs (Fabs 7-10) and a control Fab1 are shown from the 35 new Fab mutants tested in the first-round screening.

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

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

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

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

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

[0050] Figures 10A-10B show T cell activation. Figure 10A shows activation of T cell markers CD25 and CD69 on SKOV-3 cells by various low-affinity anti-CD3 Fab-HK129-folate molecules. Figure 10B shows activation of T cell markers CD25 and CD69 on SKOV-3 cells by various low-affinity anti-CD3 Fab-HK129-folate molecules.

[0051] Figures 11A-11D show the in vitro cytokine release induced by IFNγ (Figures 11A and 11B) and TNFα (Figures 11C and 11D) of various low-affinity anti-CD3 Fab-HK129-folate mutants in the absence (Figures 11A and 11C) or presence (Figures 11B and 11D) of SKOV-3 tumor cells.

[0052] Figures 12A-12F show anti-CD3 Fab-folate bispecific conjugates and PEG linkers of the invention. Figure 12A shows a representative example of a folate-PEG ligand; Figure 12B shows a representative example of a folate-branched PEG ligand; Figures 12C-12D show a representative example of a CD3-folate bispecific conjugate containing a PEG conjugate; Figure 12E shows a representative example of a CD3-folate bispecific conjugate containing C-terminal PEGylation; and Figure 12F shows a representative example of a CD3-folate bispecific conjugate containing C-terminal PEGylation via cross-linking of the CD3-Fab.

[0053] Figures 13A-13D show CD3Fab-folate conjugates with and without PEGylation. Figures 13A and 13B show SDS-PAGE gel electrophoresis analysis of anti-CD3Fab mono- and biconjugate compositions; Figure 13C shows SDS-PAGE gel electrophoresis analysis of anti-CD3Fab-folate C-terminal PEG conjugates; and Figure 13D shows the in vitro cytotoxicity of anti-CD3Fab-folate C-terminal PEG conjugates in KB, OV-90, and SKOV-3 cells.

[0054] FIG. 14 shows an anti-CD3 Fab-folate bispecific antibody that exhibits in vitro cytotoxicity and selectively kills FOLRα-expressing KB cells.

[0055] Figures 15A-15B show in vitro cytotoxicity: Figure 15A shows an anti-CD3 Fab-folate bispecific antibody selectively killing FOLRα-expressing SKOV3 cells in the presence of 20 nM folate, and Figure 15B shows selective killing in the presence of 50 nM folate.

[0056] Figures 16A-16B show in vitro cytotoxicity data demonstrating that anti-CD3 Fab-folate bispecific antibodies selectively kill FOLRα-expressing SKOV3 cells in the presence of 20 nM 5-mTHF (Figure 16A) and 50 nM 5-mTHF (Figure 16B).

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

[0058] Figures 18A-18B show that anti-CD3 Fab-folate bispecific antibodies selectively kill human M2 macrophages. Figure 18A shows in vitro macrophage cytotoxicity by a single folate containing anti-CD3 Fab-folate bispecific antibody. Arrows and numbers indicate the difference (fold) between M1 and M2 macrophages in 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 dual folate containing anti-CD3 Fab-folate bispecific antibodies. The arrows and numbers indicate the difference (fold) between M1 and M2 macrophages as 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 anti-tumor efficacy of anti-CD3-folate bispecific antibodies. Figure 19A shows tumor volume, and Figure 19B shows mass / body weight. Figures 19C and 19D show human CD45 expression and TIL induction, respectively.

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

[0061] Figures 21A-21F show the antitumor efficacy of multiple doses of CD3-folate bispecific antibody in human cervical tumors derived from the 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] 22A-22B show the anti-tumor efficacy of CD3-folate bispecific antibodies in human cervical tumors derived from the KB cell line; FIG. 22A shows tumor growth and FIG. 22B shows body weight.

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

[0064] [Definition] It is to be understood that the present invention is not limited to the particular methodology, protocols, cell lines, constructs, and agents described herein, as such may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0065] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly excludes otherwise. Thus, for example, reference to "anti-CD3 Fab" or "anti-CD3 Fab-folate antibody" is a reference to one or more of such proteins and includes equivalents thereof known to those skilled in the art. The terms "PEG-folate" or "folate-PEG" and "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] The publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the structures and methods described in the publications, which may be used in connection with the present invention. The publications discussed herein are solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate the above disclosure by virtue of prior invention or for any other reason.

[0068] The term "substantially purified" refers to an anti-CD3 Fab antibody that is substantially or essentially free from components with which the protein is normally associated or interacts, as found in the environment in which it is naturally produced (i.e., native cells) or in host cells (in the case of recombinantly produced anti-CD3 antibodies). Anti-CD3 antibodies that are substantially free of cellular material include, for example, preparations that contain 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 proteins. When the anti-CD3 antibody or variant thereof is recombinantly produced from host cells, such proteins may be present at 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 the dry weight of the cells. When an anti-CD3 antibody or variant thereof is recombinantly produced in a host cell, the periplasm and / or culture medium may contain less than about 5 g / L, less than about 4 g / L, less than about 3 g / L, less than about 2 g / L, less than about 1 g / L, less than about 750 mg / L, less than about 500 mg / L, less than about 250 mg / L, less than about 100 mg / L, less than about 50 mg / L, less than about 10 mg / L, or less than about 1 mg / L of such protein based on the dry weight of the cells. Thus, the purity of a "substantially purified" anti-CD3 antibody provided by the methods of the present invention may be greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, or greater than about 70%. Specifically, the purity may be greater than about 75%, greater than about 80%, or greater than about 85%. More specifically, the purity may be about 90% or greater, about 95% or greater, about 99% or greater, or greater, as 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 exogenous polynucleotide may be inserted by any method (e.g., direct uptake, transformation, transduction, f-mating, or other methods known in the art for producing recombinant host cells). The exogenous polynucleotide may be maintained as a non-integrated vector (e.g., a plasmid), or alternatively, may be integrated into the host genome.

[0070] An antibody is a protein that exhibits binding specificity to a specific antigen. Natural antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L chains) and two identical heavy chains (H chains). Each light chain is linked to a heavy chain by a single covalent disulfide bond. The number of disulfide bonds between heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also contains interchain disulfide bridges at regular intervals. At one end of each heavy chain is a variable domain (V H ), followed by several constant domains. At one end of each light chain is a variable domain (V L ) at one end and a constant domain at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain. The variable domain of the light chain also aligns with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0071] The term "variable" refers to the fact that certain portions of the variable domains differ significantly in sequence among antibodies. These sequence differences are responsible for the binding specificity of each particular antibody to a particular antigen. However, this variability is not evenly distributed within the variable domains of antibodies. The variability is concentrated in three regions called complementarity-determining regions (CDRs), which are present in both the light-chain and heavy-chain variable domains. The more conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy-chain and light-chain variable domains each contain four FR regions, many of which adopt a beta-sheet structure and connect the three CDRs. The CDRs form loop structures, which in some cases form part of the beta-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions. 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] The constant domain is not directly involved in binding of the antibody to an antigen but exhibits various effector functions. Antibodies or immunoglobulins can be classified into different classes depending on 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 divided 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. Of the various human immunoglobulin classes, only human IgG1, human IgG2, human IgG3, and human IgM are known to activate complement. The immunoglobulin may be selected from IgG, IgA, IgD, IgE, IgM, or fragments or modifications thereof.

[0073] In vivo antibody affinity maturation proceeds primarily through antigen selection of high-affinity antibody variants generated by somatic hypermutation. A "repertoire shift" also commonly occurs, in which the dominant germline genes for the secondary or tertiary responses are found to be distinct from those for the primary and secondary responses.

[0074] The affinity maturation process of the immune system can be reproduced by introducing mutations into antibody genes in vitro and isolating mutants with improved affinity through affinity selection. These mutant antibodies can be displayed on the surface of filamentous bacteriophage or microorganisms (e.g., Escherichia coli, yeast, etc.). Antibodies can then be selected for their affinity to the antigen or for their off-rate (Hawkins et al. J. Mol. Biol. 226:889-896 (1992)). CDR-walking mutagenesis has been used to affinity-maturate human antibodies that bind to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) (Barbas III et al. PNAS (USA) 91: 3809-3813 (1994); Yang et al. J. Mol. Biol. 254:392-403 (1995)). A similar approach has also been used to affinity mature an anti-c-erbB-2 single-chain Fv fragment (Schier et al. J. Mol. Biol. 263:551-567 (1996)). A human antibody with high affinity for the third hypervariable loop of HIV was affinity matured 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)] describes computer-assisted oligodeoxyribonucleotide-specific scanning mutagenesis. This method simultaneously searches all CDRs of the variable region gene to obtain improved mutants. An initial limited mutagenesis strategy has also been used to affinity mature a humanized antibody specific for αvβ3. In this method, mutations are introduced into all six CDR positions, and then the resulting libraries are screened to obtain the highest affinity mutants (Wu et al. PNAS (USA) 95: 6037-6-42 (1998)).Antibodies displayed on phage have been reviewed in Chiswell and McCafferty's TIBTECH 10:80-84 (1992); Rader and Barbas III's Current Opinion in Biotech. 8:503-508 (1997). In all of the aforementioned publications reporting mutant antibodies with improved affinity compared to the parent antibody, the mutant antibodies had amino acid substitutions in the CDRs.

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

[0076] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially encoded by all or part of antibody genes. 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. As used herein, antibodies include full-length antibodies and antibody fragments. They also include naturally occurring antibodies in any organism and artificially produced antibodies (e.g., mutants). The antibodies described herein may be human, humanized, engineered, nonhuman, and / or chimeric. Humanized antibodies and methods for producing such antibodies 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; and 5,766,886.) Generally, a humanized antibody comprises one or more variable domains in which the CDRs or portions thereof are derived from a non-human antibody and the framework regions or portions thereof are derived from human antibody sequences. A humanized antibody may optionally comprise at least a portion of a human constant region. In some embodiments, framework residues in a humanized antibody can be substituted with corresponding residues from the non-human antibody (the antibody from which the CDR residues were derived) to, for example, restore or improve antibody specificity or affinity. A chimeric antibody may refer to an antibody generated by combining or linking two or more antibody genes originally coded for separate antibodies. For example, a chimeric antibody can be generated by combining or linking 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 may be derived from a human or cynomolgus monkey, but is not limited thereto. In certain embodiments, the antibodies described herein may be cross-species reactive, e.g., the antibody may recognize a human antigen and a cynomolgus monkey antigen (e.g., a human / canine antibody).

[0077] The term "antibody fragment" refers to an antibody in any form other than the full-length form. Antibody fragments, as used herein, include (i) antibodies that are small portions of full-length antibodies and (ii) artificially engineered antibodies. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab')2, single-chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules with other scaffolds, and bispecific antibodies (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Unless otherwise indicated, the term "antibody(s)" as used in the specification and claims may specifically include "antibody fragment(s)." In certain embodiments, "anti-CD3 antibodies," "anti-CD3Fabs," "anti-CD3Fab antibodies," and "anti-CD3Fab variants" are antibody fragments as defined herein.

[0078] As used herein, the term "computational screening method" refers to any method for designing one or more mutations in a protein that uses a computer to evaluate the interaction energies of possible amino acid side chain substitutions with each other and / or with the rest of the protein.

[0079] As used herein, the term "full antibody" refers to the structure that constitutes the natural biological form of an antibody's H and / or L chain. In many mammals (e.g., humans and mice), this form is a tetramer, consisting of two pairs of two immunoglobulin chains (two identical pairs). Each pair contains one light chain and one heavy chain. Each light chain contains the V of an immunoglobulin. L Domain and C LEach heavy chain contains the V domain of an immunoglobulin. H Each combination contains a light chain variable region and a heavy chain variable region (V L and V H ) are responsible for binding to antigens. The constant regions (CL, Cγ1, Cγ2 and Cγ3, especially Cγ2 and Cγ3) are responsible for the effector functions of the antibody. In some mammals (e.g., camels and llamas), a full-length antibody may consist of only two heavy chains. Each heavy chain contains the V domain of an immunoglobulin. H It contains the Cγ2 domain and the Cγ3 domain.

[0080] As used herein, "immunoglobulin (Ig)" refers to a protein composed of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins may take a variety of structural forms, including, but not limited to, full length antibodies, antibody fragments, and individual domains of immunoglobulins (such as, but not limited to, V H , Cγ1, Cγ2, Cγ3, V L and C L ) are mentioned.

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

[0082] As used herein, a "mutant protein sequence" refers to a protein sequence that has one or more residues that differ in amino acid identity from another similar protein sequence. The "similar protein sequence" may be a naturally occurring wild-type protein sequence or a variant that differs from the wild-type sequence. Generally, the original sequence is referred to as a "parent sequence." The parent sequence may be a wild-type sequence or a variant sequence. For example, in a preferred embodiment of the present invention, a humanized parent sequence may be used to generate variants through computer analysis of the parent sequence.

[0083] As used herein, the "variable region" of an antibody refers to the V region of an immunoglobulin. H Domain, immunoglobulin V L domain, or V of immunoglobulin H Domains and V L "Variable region" refers to one or more polypeptides composed of domains (including variants). The term "variable region" may refer to these polypeptides in isolated form (as the region contained in an Fv fragment, scFv fragment, or larger antibody fragment), or 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 antibodies of the present invention, the terms "antigen-specific" or "specifically binds" refer to an anti-CD3 antibody that binds to a given antigen or one or more epitopes of a given 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 or target-binding sites, where a first binding site has affinity for a first antigen or epitope and a second binding site has binding affinity for a second antigen or epitope (different from the first antigen or epitope). In some embodiments, a bispecific anti-CD3 antibody has a binding site that binds to CD3 and one or more binding sites that have 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 partner that is recognized by an anti-CD3 Fab antibody. When the antigen comprises a polypeptide, the epitope can be a linear or three-dimensional amino acid sequence or shape. An epitope can also be any position on any type of antigen at which an anti-CD3 antibody binds to the antigen.

[0087] As used herein, "antigen-binding polypeptide" or "anti-CD3 Fab antibody" encompasses polypeptides and proteins that have at least the biological activity of specifically binding to a particular binding partner (e.g., an antigen). Also encompassed are CD3 analogs, CD3 isoforms, CD3 mimetics, CD3 fragments, hybrid CD3 proteins, fusion proteins, oligomers and multimers, homologs, glycosylation variants, and muteins of the above polypeptides and proteins, regardless of whether they possess similar biological activity. Furthermore, methods for synthesizing or producing these substances are not limited. Such synthesis or production methods include, but are not limited to, recombinant (whether from cDNA, genomic DNA, synthetic DNA, or other forms of nucleic acid), in vitro or in vivo microinjection of nucleic acid molecules, synthetic methods, transgenic methods, and gene activated methods. Specific examples of anti-CD3 antibodies include, but are not limited to, antibody molecules, heavy chains, light chains, variable regions, CDRs, Fabs, scFvs, non-antibody molecules with other scaffolds, ligands, receptors, peptides, or any amino acid sequence that binds to an antigen.

[0088] Antigen-binding polypeptides include pharmaceutically acceptable salts and prodrugs. Prodrugs include salts, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants, and stereoisomers of natural human anti-CD3 antibodies. Agonistic, mimetic, and antagonistic variants of natural human anti-CD3 antibodies, as well as salts and polymorphs of their polypeptide fusions, are also prodrugs. Fusions with additional amino acids at the amino terminus, carboxyl terminus, or both, are also encompassed by the term "antigen-binding polypeptide." 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 (for example, but not limited to, polyhistidine or affinity epitopes), fusions for linking anti-CD3 antibodies to other biologically active molecules, fusions with serum albumin-binding peptides, and fusions with serum proteins (such as serum albumin).

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

[0090] Naturally occurring 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 are composed of distinct domains. Each light chain has one variable domain (VL) and one constant domain (CL). Meanwhile, each heavy chain has one variable domain (VH) and three constant domains (CH). Each domain (consisting of approximately 110 amino acid residues) folds into a characteristic β-sandwich structure (immunoglobulin fold) formed by two antiparallel β-sheets. Each VL domain contains three complementarity-determining regions (CDRs 1-3), and each VH domain contains up to three complementarity-determining regions (CDRs 1-3). CDRs are loops (or turns) that connect the β-strands at one end of the variable domain. Both the light and heavy chain variable regions are typically responsible for antigen specificity (although the contributions of each chain to specificity are not necessarily equal). Antibody molecules have evolved to bind to a large number of molecules by randomizing the CDR loops.

[0091] Functional substructures of Abs can be generated by proteolysis and recombinant techniques. Abs contain Fab fragments, Fv fragments, and Fc portions. Fab fragments contain the VH-CH1 domain of the heavy chain and the VL-CL1 domain of the light chain, linked by a single interchain disulfide bond. Fv fragments contain only the VH and VL domains. The Fc portion contains the non-antigen-binding region of the antibody molecule. In some cases, even a single VH domain retains significant affinity for the antigen (Ward et al., Nature 341, 554-546, 1989). It has also been shown that certain monomeric kappa light chains specifically bind to their antigens (L. Masat et al., PNAS 91:893-896, 1994). It has also been shown that isolated light or heavy chains can retain some antigen-binding activity (Ward et al., Nature 341, 554-546, 1989).

[0092] Another functional substructure is the single-chain Fv (scFv). scFvs consist of the variable regions of immunoglobulin heavy and light chains covalently linked with a peptide linker (Sz Hu et al., Cancer Research, 56, 3055-3061, 1996). These small proteins (Mr: 25,000 Da) typically retain antigen specificity and affinity as a single polypeptide and can provide convenient building blocks for larger antigen-specific molecules. However, scFvs have short circulating half-lives, often limiting their therapeutic utility.

[0093] Small protein scaffolds called "minibodies" were designed using a portion of the VH domain of Ig as a template (Pessi et al., Nature 362, 367-369, 1993). Minibodies with high affinity for interleukin-6 (dissociation constants (K d ): approx. 10 -7 M) have been discovered by randomizing the loops corresponding to CDR1 and CDR2 of VH and then selecting the mutants by phage display (Martin et al., EMBO J. 13, 5303-5309, 1994).

[0094] Analysis of IgG-like substances from camel serum typically lacks a light chain variable domain. This suggests that sufficient antibody specificity and affinity can be derived solely from the VH domain (three or four CDR loops). High-affinity "camelized" VH domains have been produced. High specificity can also be achieved by randomizing only the CDR3.

[0095] An alternative to "minibodies" is "diabodies." Diabodies are small, bivalent, bispecific antibody fragments with two antigen-binding sites. These fragments contain heavy chain variable domains (V H ) and the light chain variable domain (VL ), which are linked together on the same polypeptide chain (V H -V L ). Diabodies are similar in size to Fab fragments. The linker in diabodies is too short to allow pairing between two domains on the same chain; therefore, each domain must pair with the corresponding domain on the other chain, thus creating two antigen-binding sites. 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 mimetics have been created (Dougall et al., 1994, Trends Biotechnol. 12, 372-379). CDR peptides are short, usually cyclic, peptides that correspond 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 mimetics have been shown to retain some binding affinity (Smyth & von Itzstein, J. Am. Chem. Soc. 116, 2725-2733, 1994). Mouse CDRs have been grafted onto human Ig scaffolds without loss of affinity (Jones et al., 1986, Nature 321, 522-525; Riechmann et al., 1988).

[0097] In humans, specific Abs are selected and amplified from large libraries (affinity maturation). This process can be replicated in vitro using combinatorial library technology. By successfully displaying Ab fragments on the surface of bacteriophage, it is possible to generate 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). An increasing number of Fabs and Fvs (and their derivatives) have been generated using this technology. Combinatorial technology can also be combined with pseudo-Ab technology.

[0098] A number of protein domains that serve as potential protein scaffolds have been expressed in fusion with phage capsid proteins (see Clackson & Wells, Trends Biotechnol. 12:173-184, 1994 for a review). Some of these protein domains have already been used as scaffolds for displaying random peptide sequences. Examples 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 streptococcal IgG-binding domain (O'Neil et al., Techniques in Protein Chemistry V (Crabb, L,. ed.) pp. 517-524, Academic Press, San Diego, 1994). These scaffolds display one randomized loop or region. Tendamistat has been used as a scaffold for display on the filamentous phage M13 (McConnell and Hoess, J. Mol. Biol. 250:460-470, 1995).

[0099] Covalent attachment of the hydrophilic polymer poly(ethylene glycol) (abbreviated as PEG) can (i) improve water solubility and bioavailability, (ii) increase serum and therapeutic half-life, (iii) modulate immunogenicity and bioactivity, or (iv) extend the circulation time of many biologically active molecules (e.g., proteins, peptides, and especially hydrophobic molecules). PEG has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, reduced toxicity, and reduced immunogenicity are important. To maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high. This can confer the typical benefits associated with the addition of PEG polymers, such as increased water solubility and increased circulatory half-life, without adversely affecting the biological activity of the parent molecule.

[0100] PEG derivatives are commonly linked to biologically active molecules via reactive chemical functionalities (e.g., lysine, cysteine, and histidine residues, the N-terminus, and carbohydrate moieties). Proteins and other molecules typically have only a limited number of reactive sites available for polymer attachment. The sites most suitable for polymer attachment typically play a key role in receptor binding and are essential for maintaining the biological activity of the molecule. Consequently, indiscriminate attachment of polymer chains to reactive sites on biologically active molecules typically results in a significant reduction or complete loss of the biological activity of the polymer-modified molecule (R. Clark et al., J. Biol. Chem., 271:21969-21977, 1996). Previous approaches to forming conjugates with sufficient polymer molecular weight to confer the desired benefit on target molecules have generally involved random attachment of multiple polymer chains to the molecule, an approach that is therefore highly likely to reduce or completely eliminate the biological activity of the parent molecule.

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

[0102] For example, the amino acid lysine has an --NH2 moiety at the ε- and α-positions. The ε-NH2 moiety is free to react under basic pH conditions. Much of the technology in the field of protein derivatization with PEG involves the development of PEG derivatives for attachment 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 common limitation: they cannot be selectively placed among the numerous lysine residues present on the surface of proteins. This can be a significant limitation when lysine residues are important for protein activity, such as those present in enzyme active sites, or when lysine residues are involved in mediating interactions between proteins and other biological molecules, such as in the binding sites of receptors.

[0103] A second, equally significant challenge with existing methods for PEGylating proteins is the potential for undesired side reactions of PEG derivatives with residues other than the desired one. Histidine has a reactive imino moiety, 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, 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, heterogeneous mixture of PEG-derivatized bioactive molecules, potentially destroying the activity of the targeted bioactive molecules. It would be desirable to develop PEG derivatives that allow for the introduction of chemical functional groups at a single site within a protein, thereby enabling the selective coupling of one or more PEG polymers to bioactive molecules at specific, well-defined sites on the protein surface.

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

[0105] As recognized in the art, many of these derivatives developed for attachment to protein side chains, particularly the --NH2 moiety on the side chain of lysine amino acids and the --SH moiety on the side chain of cysteine, have proven problematic in their synthesis and use. Some degrade through hydrolysis or form unstable bonds with proteins that are unstable in aqueous environments, such as the bloodstream. Some form more stable bonds but are hydrolyzed before the bond is formed. This means that the reactive group on the PEG derivative may be inactivated before attachment to the protein. Some are toxic to some extent and therefore less suitable for in vivo use. Some react too slowly to be practical. Some are attached to sites involved in protein activity, resulting in loss of protein activity. Some are not specific to the site to which they are attached, which can also result in loss of desired activity and lack of reproducibility of results. To overcome the challenges associated with modifying proteins with poly(ethylene glycol) moieties, PEG derivatives have been developed that are more stable (e.g., U.S. Pat. No. 6,602,498, incorporated herein by reference) or that selectively react with thiol moieties on molecules and surfaces (e.g., U.S. Pat. No. 6,610,281, incorporated herein by reference). There is a clear need in the art for PEG derivatives that are chemically inert in physiological environments until required to selectively react to form stable chemical bonds.

[0106] Recently, an entirely new technology in protein science has been reported that promises 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 allows the incorporation of non-genetically encoded amino acids into proteins in vivo. Using this method, numerous new amino acids with novel chemical, physical, or biological properties (e.g., photoaffinity-labeled and photoisomerizable amino acids, ketoamino acids, and glycosylated amino acids) have been efficiently and precisely 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 demonstrated that it is possible to selectively and routinely introduce chemical functional groups not found in proteins (e.g., ketone, alkyne, and azide moieties) that are chemically inert and can be used to react efficiently and selectively to form stable covalent bonds with all of the functional groups found in the 20 common genetically encoded amino acids.

[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 react cleanly and efficiently to form stable bonds while being inert toward the functional groups of the 20 common (genetically encoded) amino acids. For example, those skilled in the art are aware that azide and acetylene groups undergo the Huisgen [3 + 2] cycloaddition reaction under aqueous conditions in the presence of catalytic amounts of copper (see, e.g., Tornoe, et al., (2002) Org. Chem. Int. Ed. 67:3057-3064; Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599). The introduction of azide moieties into protein structures allows the incorporation of functional groups that are chemically inert toward, for example, amine, sulfhydryl, carboxylic acid, and hydroxyl groups in proteins, yet react smoothly and efficiently with acetylene moieties to form cycloaddition products. Importantly, in the absence of acetylene moieties, azides remain chemically inert and do not react in the presence of other protein side chains and under physiological conditions.

[0108] Various literature discloses modification of polypeptides by polymer conjugation or glycosylation. The term "anti-CD3 antibody" or "antigen-binding polypeptide" refers to a polypeptide that retains one or more of the biological activities of a natural antibody, in addition to the anti-CD3 antibodies described above. Such biological activities include, but are not limited to, activities other than antigen binding. Activities other than antigen binding include, but are not limited to, any one or more activities associated with Fc. The term "anti-CD3 antibody" or "antigen-binding polypeptide" also includes, but is not limited to, polypeptides conjugated to polymers such as PEG, which may additionally have one or more derivatized residues, such as cysteine, lysine, N-terminal or C-terminal amino acids, or other residues. Furthermore, anti-CD3 antibodies may have a linker, polymer, or biologically active molecule. The amino acid conjugated to the linker, polymer, or biologically active molecule may be a non-natural amino acid according to the present invention. Alternatively, the amino acid may be conjugated to a naturally encoded amino acid using known techniques (e.g., coupling with lysine or cysteine). U.S. Patent No. 4,904,584 discloses lysine-reduced PEGylated polypeptides, in which one or more lysine residues are deleted or substituted with other amino acid residues. International Publication No. WO 99 / 67291 discloses a method for conjugating a protein with PEG, in which one or more amino acid residues are deleted from the protein and the protein is contacted with PEG under conditions sufficient for protein conjugation. International Publication No. WO 99 / 03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, in which cysteine ​​residues are substituted with non-essential amino acid residues located in specific regions of the polypeptide. International Publication No. WO 00 / 26354 discloses a method for producing glycosylated polypeptide variants with reduced allergenicity, in which the polypeptide variants have one or more additional glycosylation sites compared to the corresponding parent polypeptide.

[0109] The term "antigen-binding polypeptide" also includes glycosylated anti-CD3 antibodies. Glycosylated anti-CD3 antibodies are polypeptides glycosylated at any amino acid position, including, but not limited to, N-linked or O-linked glycosylation. Variants with a single nucleotide mutation are also considered biologically active anti-CD3 antibody variants. Splicing variants are also included within the term "antigen-binding polypeptide." The term "antigen-binding polypeptide" also includes heterodimers, homodimers, heteromultimers, or homomultimers of one or more anti-CD3 antibodies. Antigen-binding polypeptides also include substances chemically linked to, or expressed as fusion proteins with, any other polypeptides, proteins, carbohydrates, polymers, small molecules, linkers, ligands, or any other type of biologically active molecule. Polypeptide analogs with, for example, specific deletions or other modifications that still retain biological activity are also included.

[0110] In some embodiments, the antigen-binding polypeptide further comprises an addition, substitution, or deletion that modulates the biological activity of the anti-CD3 antibody. For example, the addition, substitution, or deletion may modulate one or more properties or activities of the anti-CD3 antibody. Examples of such modulation include, but are not limited to, (i) modulating affinity for the antigen, (ii) modulating antigen conformation or other secondary, tertiary, or quaternary structure (e.g., increasing or decreasing the amount of change), (iii) stabilizing antigen conformation or other secondary, tertiary, or quaternary structure, (iv) inducing antigen conformation or other secondary, tertiary, or quaternary structure, (v) modulating circulating half-life, (vi) modulating therapeutic half-life, (vii) modulating polypeptide stability, (viii) modulating dosage, (ix) modulating release or bioavailability, (x) facilitating purification, and (xi) improving or altering a particular route of administration. Similarly, antigen-binding polypeptides may contain protease cleavage sequences, reactive groups, antibody binding domains (such as, but not limited to, FLAG or polyHis), other affinity sequences (such as, but not limited to, FLAG, polyHis, GST), or binding molecules (such as, but not limited to, biotin) that facilitate detection of the polypeptide (such as, but not limited to, GFP), facilitate purification, or improve other attributes.

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

[0112] One of skill in the art will appreciate that amino acid positions corresponding to positions in a particular antigen-binding polypeptide sequence can be readily identified in fragments of that antigen-binding polypeptide or related antigen-binding polypeptides, etc. For example, sequence alignment programs (such as BLAST) can be used to align and identify particular positions in a protein that correspond to positions in the related sequence.

[0113] The term "antigen-binding polypeptide" also includes antigen-binding polypeptides with one or more amino acid substitutions, additions, or deletions. Antigen-binding polypeptides of the present invention may also have modifications in which one or more naturally occurring amino acids are combined with one or more unnatural amino acids. Representative examples of substitutions at various amino acid positions in naturally occurring anti-CD3 antibody polypeptides have been described above. Examples include, but are not limited to, substitutions that modulate one or more biological activities of the antigen-binding polypeptide. Specific examples include, but are not limited to, improved agonist activity, improved solubility of the polypeptide, and conversion of the polypeptide into an antagonist. These examples are also encompassed by the term "anti-CD3 antibody."

[0114] A "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 may be used synonymously with the term "non-natural amino acid" include "non-natural amino acid," "unnatural amino acid," "non-naturally-occurring amino acid," "non-canonical amino acid," and hyphenated and non-hyphenated forms of the above. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that arise from modification (e.g., post-translational modification) of a naturally encoded amino acid (such as, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but that are not themselves normally incorporated into a growing polypeptide chain by the translation complex. Examples of such unnatural amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In some embodiments, the unnatural amino acid comprises 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 is replaced with a covalent bond not typically found in nature. Such amino acids include, but are not limited to, alkenes, oximes, thioethers, amides, and the like. Examples of such amino acids also include sugars not commonly found in natural proteins (2-deoxy-glucose, 2-deoxygalactose, etc.).Specific examples of unnatural amino acids include, but are not limited to, 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.

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

[0116] The terms "functional group," "active site," "activating group," "leaving group," "reactive site," "chemically reactive group," and "chemically reactive site" are used in the art and herein to refer to distinct and definable portions or units in a molecule. These terms generally have the same meaning in the chemical arts. They are used herein to refer to portions of molecules that have some function or activity or that react with other molecules.

[0117] As used herein, the term "linkage" or "linker" refers to a group or bond that is typically formed as a result of a chemical reaction and is typically a covalent bond. A "hydrolytically stable linkage" means that the linkage is substantially stable in water and does not react with water at practical pH values ​​(e.g., but not limited to, does not react for extended periods (possibly indefinitely) under physiological conditions). A "hydrolytically unstable linkage" or "hydrolytically degradable linkage" means that the linkage is degradable in water or aqueous solutions (e.g., blood). An "enzyme-labile linkage" or "enzyme-degradable linkage" means that the linkage can be degraded by one or more enzymes. As known in the art, PEG and related polymers may contain degradable linkages (i) in the polymer backbone or (ii) in a linker group between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, an ester linkage formed by reaction of a PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group of a biologically active agent typically hydrolyzes under physiological conditions to release the agent. Other hydrolytically degradable linkages include, but are not limited to, (i) carbonate linkages, (ii) imine linkages resulting from the reaction of an amine with an aldehyde, (iii) phosphate linkages formed by the reaction of an alcohol with a phosphate group, (iv) hydrazone linkages, which are the reaction product of a hydrazide with an aldehyde, (v) acetal linkages, which are the reaction product of an aldehyde with an alcohol, (vi) orthoester linkages, which are the reaction product of a formate with an alcohol, (vii) peptide linkages formed between an amine group (e.g., but not limited to, at the terminus of a polymer such as PEG) and a carboxy group of a peptide, and (viii) oligonucleotide linkages formed between a phosphoramidite group (e.g., but not limited to, at the terminus of a polymer) and a 5' hydroxyl group of an oligonucleotide. Branched linkers may also be used in the antigen-binding polypeptides of the present invention.

[0118] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active agent" refer to any substance capable of affecting some physical or biochemical property of a biological system, biological pathway, biological molecule, or interaction associated with an organism (e.g., but not limited to, viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans). Specifically, as used herein, "biologically active molecule" includes, but is not limited to, (i) any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or (ii) any substance intended for the improvement of the physical or mental well-being of humans or animals. Examples of biologically active 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 biologically active agents suitable for use in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytics, hormones, growth factors, steroids, and microbial toxins.

[0119] The anti-CD3 Fab-folate antibodies of the present invention may be conjugated with a molecule such as PEG to improve in vivo delivery and pharmacokinetic profile. Leong et al. have disclosed that site-specific PEGylation of the Fab' fragment of an anti-IL-8 antibody reduced the clearance rate compared to the unPEGylated form, with little or no loss of antigen-binding activity (Leong, SR et al. (2001) Cytokine 16:106-119).

[0120] Numerous other cleavable linkers are known to those of skill in the art (see U.S. Pat. Nos. 4,618,492, 4,542,225, and 4,625,014). Mechanisms for drug release from these linker groups include, for example, photoirradiation of a photolabile bond and acid-catalyzed hydrolysis. For example, U.S. Pat. No. 4,671,958 describes immunoconjugates having linkers that are cleaved at target sites in vivo by proteolytic enzymes of the patient's complement system. The length of the linker may be predetermined or may be selected depending on the desired spatial relationship between the anti-CD3 antibody and the molecule to which it is linked. Numerous methods for attaching various radioactive diagnostic compounds, radioactive therapeutic compounds, drugs, toxins, and other agents to antibodies have been reported. In light of this, one of skill in the art would be able to determine the appropriate method for attaching a given agent to an anti-CD3 antibody or other polypeptide.

[0121] A "bifunctional polymer" refers to a polymer having two distinct functional groups that can react specifically 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 on a particular biologically active component and (ii) another group capable of reacting with a group on a second biologically active component may be used to form a conjugate comprising the first biologically active component, the bifunctional linker, and the second biologically active component. Many techniques and linker molecules are known for attaching various compounds to peptides. See, e.g., 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). A "multifunctional polymer" refers to a polymer having two or more distinct functional groups that can specifically react with other moieties (such as, but not limited to, amino acid side chains) to form covalent or non-covalent bonds. Bifunctional or multifunctional polymers may be of any desired molecular length or molecular weight, and may be selected to provide desired and specific spacing or configurations between molecules linked to anti-CD3 antibodies.

[0122] As used herein, the term "water-soluble polymer" refers to any polymer that can be dissolved in an aqueous solvent. Conjugation of a water-soluble polymer to an anti-CD3 antibody can result in, but is not limited to, the following changes: (i) an increase or decrease in serum half-life or therapeutic half-life compared to the unmodified antibody; (ii) an increase in immunogenicity; (iii) an increase in physical association properties (such as aggregation and multimerization); and (iv) an increase in receptor binding and receptor dimerization or multimerization. The water-soluble polymer may or may not have biological activity itself. Alternatively, the water-soluble polymer may be used as a linker to attach another substance to the anti-CD3 antibody. The other substance may include, but is not limited to, one or more anti-CD3 antibodies or one or more biologically active molecules. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol-propionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives of polyethylene glycol (as described in U.S. Pat. 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 copolymers, polyoxyethylated polyols, 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 derivatives thereof, polyalkylene glycol copolymers and derivatives thereof, polyvinyl ethyl ether, α-β-poly[(2-hydroxyethyl)-DL-aspartamide], and the like, 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 term "polyalkylene glycol" or "poly(alkylene glycol)" refers 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 polyalkylene glycol is 0.1 kDa to 100 kDa. In some embodiments, the "polyalkylene glycol" or "poly(alkylene glycol)" may be about 5K to 50K, or within the range of 5K to 50K. For example, commercial manufacturer catalogs (such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001)) summarize other exemplary embodiments. As used herein, poly(ethylene glycol) having molecular weights of 5 kDa, 10 kDa, 20 kDa, etc., are referred to as "5K PEG," "10K PEG," "20K PEG," etc., respectively.

[0124] As used herein, the term "modulated serum half-life / modulating serum half-life" refers to a positive or negative change in the circulating half-life of a modified anti-CD3 antibody 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. Because serum concentration correlates with time, serum half-life can be calculated. While an increase in serum half-life of about two-fold or more is desirable, smaller increases in serum half-life may also be useful, for example, to provide an adequate dosing regimen or to prevent toxic effects. In some embodiments, the serum half-life is increased by about three-fold or more, about five-fold or more, about 10-fold or more, about 15-fold or more, about 20-fold or more, about 25-fold or more, about 30-fold or more, about 40-fold or more, or about 50-fold or more.

[0125] As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of (i) an anti-CD3 antibody, or (ii) an anti-CD3 antibody with a modified biologically active molecule, compared to the unmodified version. Therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various time points after administration. Increasing the therapeutic half-life may be desirable to confer a specific advantage to the dosing regimen or total dose or to avoid undesirable effects. In some embodiments, the therapeutic half-life is increased 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] The term "isolated" when applied to a nucleic acid or protein means that the nucleic acid or protein is substantially free from other cellular components with which it is naturally associated. This can be a homogeneous state. An isolated material can be dry, semi-dry, or in solution (such as, but not limited to, an aqueous solution). Purity and homogeneity are typically determined using analytical techniques (such as polyacrylamide gel electrophoresis or high-performance liquid chromatography). A protein that predominates among the species present in a preparation is substantially purified. Specifically, an isolated gene is separated from open reading frames encoding proteins other than its adjacent gene. The term "purified" means that the nucleic acid or protein appears as substantially a single band in an electrophoretic gel. Specifically, "purified" means that the nucleic acid or protein is 85% or more, 90% or more, 95% or more, 99% or more, or more pure.

[0127] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides, and polymers thereof. A "nucleic acid" may be in single-stranded or double-stranded form. Unless specifically limited, the term also includes nucleic acids containing known analogs of natural nucleotides. These analogs have binding properties equivalent to those of the nucleic acid to which they are compared and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs (e.g., PNA (peptide nucleic acid)) and DNA analogs used in antisense technology (e.g., phosphorothioates, phosphoramidates, etc.). Unless otherwise specified, a particular nucleic acid sequence substantially encompasses (i) conservatively modified variants (e.g., including, but not limited to, substitutions of degenerate codons) and (ii) complementary sequences, in addition to the sequence explicitly indicated. Specifically, substitution with degenerate codons may be performed by generating sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and / or deoxyinosine residues (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] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are not naturally encoded amino acids. As used herein, the term includes amino acid chains of any length, such as full-length proteins (i.e., antigens) in which the amino acid residues are linked by covalent peptide bonds.

[0129] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as 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 analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc.). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones. However, the same basic chemical structure as naturally occurring amino acids is preserved.

[0130] In this specification, amino acids may be represented by their 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 their commonly used one-letter symbols.

[0131] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or essentially identical amino acid sequences. Where a 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 many functionally identical nucleic acids that encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, wherever alanine is specified by a codon, that codon can be altered to any of the corresponding codons listed above without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. As used herein, every nucleic acid sequence that encodes a polypeptide also describes all possible silent variations of that nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid can be altered to yield a functionally identical molecule (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan). Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is essentially included in each described sequence.

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

[0133] The following eight groups each contain 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); and (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 "percent identity," 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. 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 specified region, optionally about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity) when compared and aligned for maximum correspondence over a measured comparison window or designated region using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. This definition also refers to the complement of a 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, polynucleotide, or polypeptide.

[0135] When comparing sequences, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

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

[0137] As used herein, the term "effective amount" refers to the amount of (modified) non-natural amino acid polypeptide administered that will relieve to some extent one or more of the symptoms of the disease, condition, or disorder being treated. Compositions containing the (modified) non-natural amino acid polypeptides described herein can be administered for prophylactic, enhancing, and / or therapeutic treatments.

[0138] The terms "enhance" or "enhancing" mean to increase or prolong, either in strength or duration, a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the term "enhancing" refers to the ability to increase or prolong, either in strength or duration, the effect of other therapeutic agents in a certain system. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of the therapeutic agent in a desired system. When used in reference to a patient, amounts effective for such use will depend on the severity and course of the disease, disorder or condition, previous treatment history, the patient's health status and response to the drugs, and the judgment of the treating physician.

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

[0140] The terms "post-translationally modified" and "modified" refer to any modification of a natural or unnatural amino acid that occurs after the amino acid is incorporated into a polypeptide chain. This term includes, by way of example only, in vivo modifications during translation, in vivo modifications after translation, and in vitro modifications after translation.

[0141] In prophylactic or therapeutic applications, compositions containing (modified) non-natural amino acid polypeptides are administered to a patient already suffering from a disease, condition, or disorder in an amount sufficient to treat (or to at least partially inhibit the symptoms of the disease, disorder, or condition). Such an amount is defined as a "prophylactically effective amount" or a "therapeutically effective amount." A prophylactically or therapeutically effective amount depends on the severity and course of the disease, disorder, or condition, previous treatment, the patient's health status and response to the drugs, and the judgment of the treating physician. It is understood in the art that such therapeutically effective amounts can be determined by routine experimentation (e.g., a dose escalation clinical trial).

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

[0143] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art, are employed.

[0144] Detailed Description of the Invention [Introduction] The present inventors have developed bispecific antibodies comprising molecules or agents that are biologically active in the absence or presence of a water-soluble polymer molecule. In a specific embodiment, the present invention provides bispecific antibodies comprising an anti-CD3 antibody, fragment, or variant comprising one or more folate molecules in the absence or presence of one or more PEG molecules. The one or more PEG molecules may be a single PEG or double PEGs, e.g., single or double 5K, 10K, 20K, or higher PEGs. The one or more PEG molecules may be linear or branched. Anti-CD3 antibodies, fragments, or variants include anti-CD3 Fab antibodies engineered to contain one or more non-naturally encoded amino acids (e.g., paraacetylphenylalanine (pAF)) at any suitable position in the heavy or light chain amino acid sequence of the Fab, including, but not limited to, positions 114, 115, 129, or 160 (Kabat numbering) in the heavy chain and positions 157, 172, or 205 (Kabat numbering) in the light chain. Bispecific antibodies can include an anti-CD3 Fab engineered to contain one or more non-naturally encoded amino acids (e.g., paraacetylphenylalanine (pAF)) on the heavy chain (K129; Kabat numbering) and light chain (L157; Kabat numbering) of the Fab. Bispecific antibodies can consist of an anti-CD3 Fab engineered to contain one or more non-naturally encoded amino acids (e.g., paraacetylphenylalanine (pAF)) on the heavy chain (K129; Kabat numbering) and light chain (L157; Kabat numbering) of the Fab. In some embodiments of the invention, one or more PEG molecules can be conjugated or linked to one or more folate molecules conjugated to one or more non-natural amino acids (e.g., pAF) and incorporated into the anti-CD3 antibody using proprietary oxime chemistry, resulting in, for example, one or two PEG molecules and / or folate molecules stably conjugated to the anti-CD3 Fab. The addition of one or more PEG molecules (e.g., 5K PEG, 10K PEG, or 20K PEG) significantly improves the pharmacokinetic properties of bispecific antibodies 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-CD3 Fab-folate compositions described herein. This result suggests that improved pharmacokinetic properties may allow for less frequent administration and avoid the need for administration via an infusion pump. The terms "PEG-folate" or "folate-PEG" and "BiPEG-BiFolate" or "BiFolate-BiPEG" are used interchangeably herein.

[0145] [Antibodies, antibody fragments, and variants thereof] The antibody, antibody fragment, or variant of the present invention may 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 may have two or more amino acid sequences. The first amino acid sequence may comprise a first antibody chain, and the second amino acid sequence may comprise a second antibody chain. The first antibody chain may have a first amino acid sequence, and the second antibody chain may have a second amino acid sequence. An antibody chain may refer to a combination of an antibody heavy chain, an antibody light chain, or a region, or all of an antibody heavy chain and region, or all of an antibody light chain. As a non-limiting example, an antibody herein includes a heavy chain, a fragment, or a variant thereof, and a light chain, a fragment, or a variant thereof. The two amino acid sequences of an antibody (including the two antibody chains) may be linked by one or more disulfide bonds, chemical linkers, peptide linkers, or a combination thereof. Chemical linkers include linkers via unnatural amino acids. Chemical linkers include linkers mediated by one or more unnatural amino acids. Chemical linkers can include chemical conjugates. Peptide linkers can have any amino acid sequence that connects two amino acid sequences. Peptide linkers can contain 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, or 100 or more amino acids. Peptide linkers can be any portion of an antibody, including antibody domains such as the variable domain, CH1, CH2, CH3, and / or CL domain. In some embodiments, the heavy and light chains are linked, for example, via a peptide linker. In some examples, the heavy and light chains are linked, for example, by one or more disulfide bonds.

[0146] The antibodies, antibody fragments, and antibody variants described herein may interact with or engage antigens on effector cells. Effector cells include, but are not limited to, immune cells, genetically modified cells with increased or decreased cytotoxic activity, cells involved in host defense mechanisms, anti-inflammatory cells, leukocytes, lymphocytes, macrophages, erythrocytes, platelets, neutrophils, monocytes, eosinophils, basophils, mast cells, NK cells, B cells, or T cells. In some embodiments, the immune cells may be T cells (cytotoxic T cells or natural killer T cells). The antibodies or antibody fragments may interact with receptors on T cells, including, but not limited to, T cell receptors (TCRs). The TCRs may include TCRα, TCRβ, TCRγ, and / or TCRδ or TCRζ. The antibodies or antibody fragments of the present invention may 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 may bind to cell surface receptors. The antibody or antibody fragment of the present invention can bind to a folate receptor. The antibody or antibody fragment of the present invention can be conjugated to a T cell surface antigen (for example, but not limited to, 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) or its analogs or derivatives). 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, antibody fragments, or variants described herein may be humanized. The anti-CD3 antibodies, 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), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules with other scaffolds, bispecific antibodies, and the like. The anti-CD3 antibodies, antibody fragments, or variants of the present invention have the sequences of SEQ ID NOs: 1-62. 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 comprise one or more anti-CD3 Fabs. The antibody, fragment, or variant of the present invention may comprise two anti-CD3 Fabs. In certain embodiments, the anti-CD3 antibody has a heavy 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 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 a heavy chain amino acid sequence of 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 a light chain amino acid sequence of any one of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62.

[0148] Anti-CD3 bispecific antibodies comprising unnatural 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), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, non-antibody molecules with other scaffolds, bispecific antibodies, etc. In some embodiments, the anti-CD3 bispecific antibody, antibody fragment, or variant is an anti-CD3 Fab bispecific antibody, fragment, or variant comprising one or more non-naturally encoded amino acids. The anti-CD3 Fab bispecific antibody, antibody fragment, or variant of the invention can have one or more of the sequences set forth in SEQ ID NOs: 1-62. The bispecific antibody, fragment, or variant of the invention can also be an anti-CD3 Fab antibody, fragment, or variant. The anti-CD3 bispecific antibody can have a heavy and / or light chain amino acid sequence selected from the sequences set forth in 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 to 62. In certain embodiments, the anti-CD3 bispecific antibody has a heavy chain amino acid sequence of 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 a light chain amino acid sequence of any one 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 cross-species reactive. The bispecific antibodies, fragments, or variants may be cross-species reactive with human and monkey antigens. In some embodiments, the antibodies comprise cross-species reactive CDRs. The bispecific antibodies may be humanized antibodies.

[0149] [Table 1]

[0150] JPEG0007759805000002.jpg217164

[0151] JPEG0007759805000003.jpg64164

[0152] JPEG0007759805000004.jpg146164

[0153] JPEG0007759805000005.jpg208164

[0154] JPEG0007759805000006.jpg208164

[0155] JPEG0007759805000007.jpg218164

[0156] JPEG0007759805000008.jpg171164

[0157] [Unnatural Amino Acids] The present invention provides anti-CD3 antibodies, antibody fragments, or variants comprising one or more non-naturally encoded amino acids. The introduction of one or more non-naturally encoded amino acids into an anti-CD3 antibody may allow for the application of conjugation chemistries that involve specific chemical reactions with one or more non-naturally encoded amino acids while not reacting with the 20 common amino acids.

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

[0159] The one or more unnatural amino acids include, but are not limited to, p-azidophenylalanine (pAz), p-benzoylphenylalanine (pBpF), p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF), p-cyanophenylalanine (pCNF), p-carboxylmethylphenylalanine (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 unnatural 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, unnatural amino acids include: (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, and p-isopropyl-L-phenylalanine); (2) amino acids with aryl azide and benzophenone groups that can be photocrosslinked; (3) amino acids with inherent 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, and p-(3-oxobutanoyl)-L-phenylalanine); and (4) amino acids that have been shown to be highly reactive by X-ray crystallography. Examples of suitable non-natural amino acids include, but are not limited to, heavy atom-containing amino acids for phasing in the synthesis of phospholipids (e.g., p-iodo- and p-bromo-L-phenylalanine); (5) the redox-active amino acid dihydroxy-L-phenylalanine; (6) glycosylated amino acids (e.g., bN-acetylglucosamine-O-serine and aN-acetylgalactosamine-O-threonine); (7) fluorescent amino acids with naphthyl, dansyl, and 7-aminocoumarin side chains; (8) photocleavable and photoisomerizable amino acids with azobenzene and nitrobenzyl Cys, Ser, and Tyr side chains; (9) p-carboxymethyl-L-phenylalanine, a phosphotyrosine mimetic; (10) homoglutamine, 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, or O-phosphotyrosine. In some embodiments, the unnatural amino acid comprises 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 is replaced with a covalent bond not typically 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 not typically found in natural proteins (2-deoxy-glucose, 2-deoxygalactose, etc.). Specific examples of unnatural amino acids include, but are not limited to, 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. Additional unnatural amino acids are described 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 Nos. 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 incorporated by reference in its entirety). In some embodiments, the one or more unnatural amino acids can be p-acetylphenylalanine (pAF).

[0160] In certain embodiments of the invention, anti-CD3 antibodies bearing one or more unnatural amino acids comprise one or more post-translational modifications. In one embodiment, the one or more post-translational modifications include the addition of a molecule, including, but not limited to, a water-soluble polymer, a polyethylene glycol derivative, a drug, a second protein or polypeptide or polypeptide analog, an antibody or antibody fragment, a biologically active agent, a small molecule, or any combination of the above, or any other desired compound or substance. These molecules contain a second reactive group that reacts with the one or more unnatural amino acids bearing the first reactive group. This reaction occurs using chemical methods known to those skilled in the art to be appropriate for the particular reactive group. For example, the first reactive group is an alkynyl moiety (such as, but not limited to, in the unnatural amino acid p-propargyloxyphenylalanine (the propargyl group is sometimes referred to as an acetylene moiety)) and the second reactive group is an azide moiety. In this example, a [3+2] cycloaddition chemistry is utilized. In another example, the first reactive group is an azido moiety (e.g., but not limited to, in the unnatural amino acid p-azido-L-phenylalanine) and the second reactive group is an alkynyl moiety. Certain embodiments of the modified anti-CD3 antibody polypeptides of the invention utilize one or more unnatural amino acids with one or more post-translational modifications (e.g., but not limited to, unnatural amino acids with a keto functional group) in the sugar moiety of the one or more post-translational modifications. In certain embodiments, the post-translational modifications are made in vivo, in a eukaryotic or non-eukaryotic cell. In other embodiments, the post-translational modifications are made in vitro. In other embodiments, the post-translational modifications are made in vitro and in vivo.

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

[0162] In some embodiments described herein, unnatural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. In some embodiments, unnatural amino acids are site-specifically incorporated into anti-CD3 antibodies, antibody fragments, or variants. Methods for incorporating unnatural amino acids into molecules (e.g., proteins, polypeptides, or peptides) 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 of which is incorporated by reference in its entirety), as well as in the Examples herein. One or more unnatural amino acids can be incorporated by methods known in the art. For example, cell-based or cell-free systems can be used, and auxotrophic strains can be used in place of engineered tRNAs and synthetases. In certain embodiments, orthogonal tRNA synthetases are used, e.g., as disclosed in PCT / US2002 / 012465; PCT / US2002 / 012635; PCT / US2003 / 032576; PCT / US2005 / 044041; PCT / US2005 / 043603; PCT / US2005 / 046618 (each of which is incorporated by reference in its entirety). Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include 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 include mutating one or more nucleotides in the nucleotide sequence encoding the antibody or antibody fragment or variant. Mutating one or more nucleotides in a nucleotide sequence encoding an antibody or antibody fragment or variant can include changing a codon encoding an amino acid to a nonsense codon. Incorporating one or more unnatural amino acids into an antibody or antibody fragment or variant can include 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, antibody fragment, or variant in response to an amber codon. One or more unnatural amino acids can be site-specifically incorporated into an antibody, antibody fragment, or variant. Incorporation of one or more unnatural amino acids into an antibody, antibody fragment, or variant can include one or more genetically encoded unnatural amino acids with chemical reactivity orthogonal to the 20 standard amino acids for site-specific modification of a biologically active molecule or targeted agent. Incorporation of one or more unnatural amino acids can include the use of a tRNA / aminoacyl-tRNA synthetase pair to site-specifically incorporate one or more unnatural amino acids into a biologically active molecule or targeted agent at a defined site in response to one or more amber nonsense codons. Additional methods for incorporating unnatural amino acids include, but are not limited to, those 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 produced by 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 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 involved in the chemical reaction to form a linker.The chemical reaction for forming the linker can include bioorthogonal reaction. The chemical reaction for forming the linker can include click chemistry. See, for example, International Publication No. WO2006 / 050262 (incorporated herein by reference in its entirety).

[0163] [Biologically active molecules / drugs] Described herein are anti-CD3 antibodies or antibody fragments or variants thereof, including biologically active molecules linked to the antibodies or fragments or variants via unnatural amino acids. Biologically active molecules include, but are not limited to, small molecules or drugs, non-peptide compounds, drugs, second proteins or polypeptides or polypeptide analogs or derivatives, antibodies or antibody fragments or variants, second biologically active agents, targeting agents, or any combination of the above or any other desired compounds or substances. In some embodiments, the biologically active agent is involved in the recruitment of cytotoxic T cells to cells (including, but not limited to, cancer cells or tumor cells). In some embodiments, the biologically active molecule is a small molecule, such as, but not limited to, folic acid or a derivative or analog thereof, or 2-[3-(1,3-dicarboxypropyl)ureidol]pentanedioic acid (DUPA) or a derivative or analog thereof. In some embodiments, the biologically active molecule is folate or a derivative or analog thereof. The biologically active molecule may be selected from a cell-targeting molecule, a ligand, a protein, a peptide, a peptoid, a DNA aptamer, a peptide nucleic acid, a vitamin, a substrate or substrate analog, a cholecystokinin B receptor, a gonadotropin-releasing hormone receptor, a somatostatin receptor 2, an avb3 integrin, a gastrin-releasing peptide receptor, a neurokinin 1 receptor, a melanocortin 1 receptor, a neurotensin receptor, a neuropeptide Y receptor, and a C-type lectin-like molecule 1, a receptor, a co-receptor, a transmembrane protein, or a cell marker or cell surface protein. The biologically active molecule may bind to a target cell. The biologically active molecule may bind to a cell surface protein, a cell surface marker, or a cell surface molecule on a cell. The biologically active molecule may bind to a cell surface molecule on a cell (e.g., but not limited to, a cancer cell, a tumor cell, or an immunosuppressive cell). The cell surface molecule may be a folate receptor molecule. The biologically active molecule may be an agent that binds to prostate-specific membrane antigen (PSMA), such as, but not limited to, DUPA or an analog or derivative thereof.A biologically active molecule or agent may bind to cells that over- or highly express a cell surface marker, protein or receptor.

[0164] In some embodiments, the biologically active molecule can be a folate or folic acid ligand, or an analog or derivative thereof. The biologically active molecule can bind to a folate receptor protein (FR). Such a biologically active molecule 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 folic acid-based moiety that preserves FR binding. The folic acid analog can preserve a significant portion of the structure of folic acid. Furthermore, the folic acid analog can be a slightly modified form of folic acid due to conjugation with a linker or antibody or antibody fragment or variant. For example, the folic acid analog can be slightly modified due to conjugation of the folic acid carboxy group to a linker or antibody or antibody fragment or variant. Furthermore, the folic acid can be slightly modified due to conjugation with a linker or antibody or antibody fragment, but maintain its FR binding properties. In some embodiments, the folic acid molecule targets the folate receptor alpha. In some embodiments, the folic acid molecule targets folate receptor β. In some embodiments, folic acid or folate salts are used as biologically active molecules or targeting agents to bind to folate receptor (FR) antigens that are overexpressed or highly expressed on FR+ cell lines. In some embodiments, the cells are, but are not limited to, cancer cells or immunosuppressive cells.

[0165] Biologically active molecules or agents can be site-specifically linked to one or more unnatural amino acids of an antibody, antibody fragment, or variant via one or more linkers. The linkers can be chemical linkers, peptide linkers, or a combination thereof. Chemical linkers include linkers via unnatural amino acids. Chemical linkers include linkers via one or more unnatural amino acids. Chemical linkers can include chemical conjugates. Peptide linkers can have any amino acid sequence that connects two amino acid sequences. Peptide linkers can contain 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, or 100 or more amino acids. The peptide linker may be any portion of an antibody, including antibody domains such as the variable domain, CH1, CH2, CH3, and / or CL domain. The antibody, antibody fragment, or variant may be conjugated to a biologically active molecule. The antibody, antibody fragment, or variant may be conjugated to a biologically active molecule via a chemical and / or peptide linker. In some embodiments, the invention provides an anti-CD3 antibody, antibody fragment, or variant conjugated to one or more biologically active molecules or agents. In some embodiments, the one or more biologically active molecules or agents are one or more small molecules. In some embodiments, the invention provides an anti-CD3Fab antibody, antibody fragment, or variant conjugated to one or more small molecules. In some embodiments, the invention provides an anti-CD3Fab antibody, antibody fragment, or variant conjugated to one or more folate molecules. In some embodiments, the invention provides an anti-CD3Fab antibody, antibody fragment, or variant conjugated to one or more DUPA molecules. In some embodiments, one or more folate molecules and / or one or more DUPA molecules may be conjugated to an anti-CD3Fab antibody via a chemical and / or 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 is not reactive with the 20 common amino acids. The linker or polymer may be a bifunctional linker or polymer. A bifunctional linker or polymer is a branched linker or polymer. One or more bonds involved in the linking of the anti-CD3 antibody or antigen-binding polypeptide to the biologically active molecule via a linker, polymer, or covalent bond may be irreversible, reversible, or labile under desired circumstances. One or more bonds involved in the linking 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. Various small molecules may be produced by those skilled in the art by chemical means, isolated as natural products, or produced by other means.

[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). The anti-CD3 antibodies or antibody fragments or variants comprising a non-naturally encoded amino acid can be linked to two water soluble polymers (such as two polyethylene glycol (PEG) molecules or ligands). The antibodies or antibody fragments or variants comprising a non-naturally encoded amino acid and one or more biologically active molecules can be linked to one or more water soluble polymers (such as polyethylene glycol (PEG) molecules or linkers). In some embodiments, the antibodies or antibody fragments or variants comprising a non-naturally encoded amino acid and two biologically active molecules are linked to two water soluble polymers (such as polyethylene glycol (PEG) molecules or linkers).

[0168] The method can include linking an antibody or antibody fragment to a biologically active molecule, or a water-soluble polymer, or a conjugate comprising a biologically active molecule and a water-soluble polymer. The method can include conjugating one or more linkers to the biologically active molecule to form a biologically active molecule-linker intermediate and conjugating the intermediate to the antibody or antibody fragment. The method can include conjugating one or more linkers to a PEG molecule to form a PEG-linker intermediate and conjugating the PEG-linker intermediate to the antibody or antibody fragment. The method can include conjugating one or more linkers to the antibody or antibody fragment to form an antibody-linker intermediate or antibody fragment-linker intermediate and conjugating the antibody-linker intermediate or antibody fragment-linker intermediate to another biologically active molecule, a water-soluble polymer, or a conjugate comprising a biologically active molecule and a water-soluble polymer. The methods described herein can include conjugating one or more linkers to one or more antibodies or antibody fragments, one or more biologically active molecules, or combinations thereof to generate one or more intermediates (such as antibody-linker intermediates, antibody fragment-linker intermediates, and / or biologically active molecule antibody conjugate-linker intermediates). The method can include conjugating a first linker to an antibody or antibody fragment to generate an antibody-linker intermediate or an antibody fragment-linker intermediate. The method can include conjugating the linker to the biologically active molecule to generate a biologically active molecule-linker intermediate.

[0169] Methods for producing bispecific anti-CD3 antibody conjugates of the invention can include (a) conjugating a first linker to an antibody or antibody fragment comprising one or more unnatural amino acids incorporated into the antibody or antibody fragment, (b) conjugating a second linker to a biologically active molecule to form a biologically active molecule-linker intermediate, and (c) linking the two intermediates to form an anti-CD3 antibody-biologically active molecule conjugate, where the biologically active molecule can be a small molecule (e.g., but not limited to, a folate molecule or a DUPA molecule, or an analog or derivative thereof). In certain embodiments, methods for producing bispecific anti-CD3 antibody conjugates of the invention can include (a) conjugating a first linker to an antibody or antibody fragment comprising one or more unnatural amino acids incorporated into the antibody or antibody fragment to form an antibody-linker intermediate or an antibody fragment-linker intermediate; (b) conjugating a second linker to a biologically active molecule to form a biologically active molecule-linker intermediate; and (c) linking the two intermediates to form an anti-CD3 antibody-biologically active molecule conjugate, where the biologically active molecule can be a small molecule (e.g., but not limited to, a folate molecule or a DUPA molecule, or an analog or derivative thereof). A method for producing a bispecific anti-CD3 Fab antibody-folate conjugate of the present invention can include (a) conjugating a first linker to an antibody or antibody fragment comprising one or more unnatural amino acids incorporated into the anti-CD3 Fab antibody or antibody fragment to form an antibody-linker intermediate or an antibody fragment-linker intermediate, (b) conjugating a second linker to a biologically active molecule to form a biologically active molecule-linker intermediate, and (c) linking the two intermediates to form an anti-CD3 Fab antibody-biologically active molecule conjugate, wherein the biologically active molecule comprises one or more folate molecules or DUPA molecules or analogs or derivatives, and the linker comprises one or more chemical linkers and / or peptide linkers, and the linker comprises 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 molecules is 5 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, or greater. The average molecular weight of the PEG molecules is 5K, 10K, or 20K.

[0170] Conjugation of an antibody or antibody fragment, or a biologically active molecule to one or more linkers can occur simultaneously. Conjugation of an antibody or antibody fragment, or a biologically active molecule to one or more linkers can occur sequentially. Conjugation of an antibody or antibody fragment, or a biologically active molecule to one or more linkers can occur in a single-step process (e.g., enzyme conjugation, or a chemical step, process, or reaction, etc.). Conjugation of an antibody or antibody fragment, or a biologically active molecule to one or more linkers can occur in a two-step process (e.g., two enzyme conjugations, or two chemical steps, or two processes, or two reactions, etc.). Conjugation of an antibody or antibody fragment, or a biologically active molecule to one or more linkers can occur in a two or more step process (e.g., two or more enzyme conjugations, or chemical steps, process, or reaction, etc.).

[0171] Conjugating the intermediate to an antibody or antibody fragment, or a biologically active molecule, or a water-soluble polymer can involve oxime chemistry and / or click chemistry to form an oxime bond, as is well known to those skilled in the art. The antibody or antibody fragment can contain one or more unnatural amino acids. Linking the antibody or antibody fragment to the intermediate can involve forming an oxime between the unnatural amino acid and the linker intermediate. Conjugating the linker to the antibody or antibody fragment, or the biologically active molecule or water-soluble molecule can involve ionic, covalent, or non-covalent bonding, or a combination thereof, between the linker and the antibody or antibody fragment, or the biologically active molecule or water-soluble molecule. Conjugation of antibodies or antibody fragments, or biologically active molecules or water-soluble molecules to linkers is known in the art. See, for example, Roberts et al., Advanced Drug Delivery Reviews 54:459-476 (2002).

[0172] Conjugating one or more linkers to an antibody or antibody fragment and / or biologically active molecule may include forming one or more oximes between the linker and the antibody or antibody fragment or biologically active molecule. Conjugating one or more linkers to an antibody or antibody fragment and / or biologically active molecule may include forming one or more stable bonds between the linker and the antibody or antibody fragment or biologically active molecule. Conjugating one or more linkers to an antibody or antibody fragment and / or biologically active molecule may include forming one or more covalent bonds between the linker and the antibody or antibody fragment or biologically active molecule. Conjugating one or more linkers to an antibody or antibody fragment and / or biologically active molecule may include forming one or more non-covalent bonds between the linker and the antibody, antibody fragment or biologically active molecule. Conjugating one or more linkers to an antibody or antibody fragment and / or ligand may include forming one or more ionic bonds between the linker and the antibody or antibody fragment or biologically active molecule.

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

[0174] One or more PEGs or linkers may contain a disulfide bridge linking two cysteine ​​residues using conjugation chemistry known to those skilled in the art. (See also, e.g., ThioBridge™ technology, Abzena). Two or more PEG molecules or linkers may contain a maleimide bridge linking 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 may contain a maleimide bridge linking 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 may be C-terminal PEG conjugates. In some embodiments, the C-terminal PEG molecule may not involve a covalent disulfide bond or attachment between the heavy and light chain antibodies or between antibody fragments. In some embodiments, two or more C-terminally located PEG molecules may be covalently attached or cross-linked between the heavy and light chains of an antibody or antibody fragment. Such PEG conjugates or linkers are described herein.

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

[0176] In certain embodiments described herein, the anti-CD3 Fab-folate PEGylated conjugate comprises one or more PEG molecules. In certain embodiments described herein, the anti-CD3 Fab-folate 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 attachment between the heavy chain antibody or antibody fragment and the light chain antibody or antibody fragment. In some embodiments, the C-terminal PEG molecule may be attached separately to the heavy and light chains of the antibody or antibody fragment. In some embodiments, two or more C-terminal PEG molecules may be covalently attached or cross-linked between the heavy and light chains of the antibody or antibody fragment. In some embodiments, two or more PEG molecules may be covalently attached or cross-linked via a maleimide bridge linking two cysteine ​​residues.

[0177] PEGylation can be used to improve pharmacokinetics and modulate the cytotoxicity of compositions. Because the PEG moiety adds significant hydrodynamic radius to proteins, PEGylation of proteins can increase their serum half-life by slowing renal clearance. Covalent attachment of the hydrophilic polymer poly(ethylene glycol) (abbreviated PEG) can (i) improve water solubility and bioavailability, (ii) increase serum and therapeutic half-life, (iii) modulate immunogenicity and biological activity, or (iv) extend the circulation time of many biologically active molecules (e.g., proteins, peptides, and especially hydrophobic molecules). PEG has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, elimination of toxicity, and elimination of immunogenicity are important. Preferably, PEGylation does not alter or only minimally alters the activity of biologically active molecules. Preferably, the increase in half-life is greater than any decrease in biological activity. [Rader et al. in Proc Natl Acad Sci USA. 2003 Apr 29;100(9):5396-400] discloses a method for conferring effector functions and extending serum half-lives to synthetic small molecules by reacting them with generic antibody molecules (incorporated herein by reference). The conjugate disclosed therein was created by reversible covalent conjugation between mAb 38C2 (a catalytic antibody mimicking the natural aldolase enzyme) and a diketone derivative of an integrin-targeting Arg-Gly-Asp peptidomimetic via a reactive lysine residue on the antibody. Not only was the half-life of the peptidomimetic extended, but the conjugate also possessed the ability to bind to integrin α. v β3 and α v Antibody target switching was observed to selectively target the surface of cells expressing β5.

[0178] In some embodiments, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a water soluble polymer (e.g., polyethylene glycol (PEG)) via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a folate via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody comprising 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 comprising a non-naturally encoded amino acid is linked to a water soluble polymer (e.g., polyethylene glycol (PEG)) via the side chain of the non-naturally encoded amino acid. In some embodiments, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a water soluble polymer derivative (e.g., 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, wherein an anti-CD3 antibody comprising one or more non-naturally encoded amino acids is linked to the folate, and / or to the water soluble polymer, and / or to the 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 those shown in Figures 12A-12F:

[0180] [ka]

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

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

[0184] [ka]

[0185] During the ceremony, A has the following structure:

[0186] [ka]

[0187] B is a divalent group linking 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-SS-(CH2) n’ -NHC(O)-(alkylene-O) n’ -Alkylene-, -(alkylene-O) n’ -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n’ -alkylene-U-alkylene-, where n' is independently an integer greater than or equal to 1; D is a trivalent group connecting C, F, and E; F is a water-soluble polymer such as polyethylene glycol (PEG); 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] In some embodiments, B is a substituted divalent heterohydrocarbyl residue. In some embodiments, the substituents include one or more carboxyl, ketone, and / or amide functional groups. In some embodiments, the heteroatoms are selected from N, O, and S.

[0189] In some embodiments, B has the structure:

[0190] [ka]

[0191] In some embodiments, D is a substituted trivalent heterohydrocarbyl residue. In some embodiments, the substituents include one or more carboxyl, ketone, and / or amide functional groups. In some embodiments, the heteroatoms are selected from N, O, and S.

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

[0193] [ka]

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

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

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

[0197] [ka]

[0198] wherein n is 2 to 10,000. In some embodiments, n is selected so 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-folate linker has the following structure:

[0200] [ka]

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

[0202] [ka]

[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 the unnatural amino acid.

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

[0205] [ka]

[0206] During the ceremony, J is optional 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 an optional constituent, and when present, is selected from the group consisting of 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 a linker selected from the group consisting of N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R')=NN=, -C(R')-N=N-, and -C(R')-N(R')-N(R')- (wherein each R' is independently H, alkyl, or substituted alkyl); 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 are anti-CD3 antibodies; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl.

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

[0208] [ka]

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

[0210] [ka]

[0211] In the formula, 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, polyalkyleneoxide, substituted polyalkyleneoxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene)-C(O)SR", -(alkylene or substituted alkylene)-SS-(aryl or substituted aryl), -C(O)R", -C(O)R", 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, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; Each Z1 is a bond, and CR 17 R 17 ,O,S,NR',CR 17 R 17 -CR 17 R 17 , C.R. 17 R 17 -O, O-CR 17 R17 , C.R. 17 R 17 -S, S-CR 17 R 17 , C.R. 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 a 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] [ka]

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

[0214] [ka]

[0215] wherein (a) indicates the bond to the B group, and (b) indicates the bond to the respective position within the heterocyclic group; M4 has the following structure:

[0216] [ka]

[0217] wherein (a) indicates the bond to the B group, and (b) indicates the bond to the respective position within the heterocyclic group; Each R 19 are 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, alkyl halide, alkylamine, alkylsulfonic acid, alkylnitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; Each R 16 are 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 involves the selective incorporation of non-genetically encoded amino acids, including, but not limited to, amino acids bearing functional groups or substituents (e.g., but not limited to, ketone, azide, or acetylene moieties) not found among the 20 amino acids typically incorporated into proteins in response to selective codons. The resulting amino acid is then modified by appropriate reaction with a PEG derivative. Once incorporated, the amino acid side chain can then be modified to the specific functional group or substituent found in naturally encoded amino acids using appropriate chemical methods known to those skilled in the art. A wide variety of known chemical methods are suitable for use in the present invention to incorporate water-soluble polymers into proteins. Such methods include, but are not limited to, the Huisgen [3+2] cycloaddition reaction between acetylene derivatives or azide derivatives, respectively (see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, Ed. Trost, BM, Pergamon, Oxford, pp. 1069-1109, 1991; Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, Ed. Padwa, A., Wiley, New York, pp. 1-176, 1984).

[0219] The Huisgen [3 + 2] cycloaddition method involves cycloaddition rather than nucleophilic substitution, allowing proteins to be modified with exceptional selectivity. This reaction can occur under aqueous conditions at room temperature with excellent regiospecificity (1,4 > 1,5) by adding a catalytic amount of Cu(I) salt to the reaction mixture (see, e.g., 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 proteins of the invention via [3 + 2] cycloaddition include virtually any molecule bearing an appropriate functional group or substituent, including, but not limited to, an azide or acetylene derivative. These molecules can be added to unnatural amino acids with an acetylene group (such as, but not limited to, p-propargyloxyphenylalanine) or an azide group (such as, but not limited to, p-azido-phenylalanine), respectively.

[0220] The five-membered ring resulting from the Huisgen [3 + 2] cycloaddition is generally irreversible in reducing environments and is stable to hydrolysis in aqueous environments for extended periods. Therefore, the physical and chemical properties of a wide variety of materials can be modified with the activated PEG or PEG derivatives of the present invention, even in harsh aqueous environments. More importantly, because the azide and acetylene moieties are specific for each other (and do not react with, for example, any of the 20 common genetically encoded amino acids), proteins can be modified with exceptional selectivity at one or more specific sites.

[0221] The present invention also provides water-soluble, hydrolytically stable derivatives of PEG derivatives and related hydrophilic polymers containing one or more acetylene or azide moieties. PEG polymer derivatives containing acetylene moieties couple with high selectivity to azide moieties selectively introduced into proteins in response to selectable codons. Similarly, PEG polymer derivatives containing azide moieties couple with high selectivity to acetylene moieties selectively introduced into proteins in response to selectable codons.

[0222] More specifically, the azide moiety includes, but is not limited to, alkyl azides, aryl azides, and derivatives of these azides. The alkyl azide and aryl azide derivatives may have other substituents as long as the acetylene-specific reactivity is preserved. The acetylene moiety includes alkyl acetylenes and aryl acetylenes, and derivatives thereof. The alkyl acetylene and aryl acetylene derivatives may have other substituents as long as the azide-specific reactivity is preserved.

[0223] Therefore, the term "anti-CD3 antibody" is intended to encompass 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 with an unnatural amino acid (such as, but not limited to, p-(propargyloxy)-phenylalanine) is provided. Also provided are various compositions comprising p-(propargyloxy)-phenylalanine and, further, proteins and / or cells. In one aspect, a composition comprising the unnatural amino acid p-(propargyloxy)-phenylalanine further comprises an orthogonal tRNA. The unnatural amino acid can be linked to the orthogonal tRNA (for example, but not limited to, via a covalent bond). Examples of this linkage include, but are not limited to, (i) covalent linkage to the orthogonal tRNA via an amino-acyl bond; (ii) covalent linkage to the 3' or 2' OH of the ribose sugar at the end of the orthogonal tRNA; etc.

[0225] [Measurement of the activity and affinity of anti-CD3 antibodies for their antigens or binding targets] The activity of anti-CD3 antibodies can be measured by standard in vitro or in vivo assays. For example, cells or cell lines that bind to anti-CD3 antibodies (e.g., but not limited to, cells containing the natural anti-CD3 antibody antigen or binding partner, or cells recombinantly producing the anti-CD3 antibody antigen or binding partner) can be used to assess anti-CD3 antibody binding. For non-PEGylated or PEGylated antigen-binding polypeptides with unnatural amino acids, the affinity of the anti-CD3 antibody for its antigen or binding partner can be measured using known techniques (e.g., BIAcore™ biosensor (Pharmacia) or Octet (ForteBio)).

[0226] Regardless of the method used to generate an anti-CD3 antibody, the anti-CD3 antibody will be subjected to an assay to evaluate its biological activity. Where appropriate, a tritiated thymidine assay may be performed to determine the extent of cell division. However, other biological assays may also be used to confirm the desired activity. For example, biological assays measuring the ability to inhibit the biological activity of an antigen (e.g., enzymatic, proliferative, or metabolic activity) may also indicate the activity of an anti-CD3 antibody. 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, testing for biological activity should provide an analysis of the desired outcome, such as 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), receptor affinity analysis, conformational or structural changes, or serum half-life analysis. Those skilled in the art will recognize other assays useful for testing the desired end result.

[0227] [Measurement of efficacy, in vivo functional half-life, and pharmacokinetic parameters] An important aspect of the present invention is the extended biological half-life achieved by constructing anti-CD3 antibodies, with or without conjugation of the anti-CD3 antibody to a water-soluble polymer moiety. Because serum concentrations of anti-CD3 antibodies decline rapidly, it has become important to evaluate the biological response to treatment with conjugated or unconjugated anti-CD3 antibodies and variants thereof. Preferably, the conjugated or unconjugated anti-CD3 antibodies and variants thereof of the present invention have extended serum half-lives, even after intravenous administration, allowing for measurement, for example, by ELISA or primary screening assays. Measurement of in vivo biological half-life is performed as described herein.

[0228] Pharmacokinetic parameters of antigen-binding polypeptides with non-naturally encoded amino acids can be assessed in conventional male Sprague-Dawley rats. Pharmacokinetic data for anti-CD3 antibodies have been well studied in several species and can be directly compared with data obtained for anti-CD3 antibodies with non-naturally encoded amino acids.

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

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

[0231] Therapeutic compositions containing one or more polypeptides of the invention can optionally be tested in one or more appropriate in vitro and / or in vivo animal models of disease to confirm efficacy and metabolism in tissues and to assess dosage. This testing follows methods well known in the art. Specifically, dosage can be initially determined by activity, stability, or other appropriate measure (i.e., comparative assay) of the unnatural amino acid herein to a natural amino acid homolog (e.g., including, but not limited to, a comparison of an anti-CD3 antibody modified with one or more unnatural amino acids to an anti-CD3 antibody composed of natural amino acids).

[0232] Administration can be by any route normally used for introducing a molecule into ultimate contact with blood or tissue cells. The unnatural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods for administering the polypeptides of the invention to a patient are available. Also, certain routes can typically produce a more immediate or effective effect or response than others (although more than one route can be used to administer a particular composition).

[0233] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer that composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

[0234] Polypeptide compositions can be administered by a variety of routes, including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal administration. Compositions containing non-natural amino acid polypeptides (modified or unmodified) can also be administered using liposomes. Such routes of administration and appropriate formulations are well known to those of skill in the art.

[0235] Anti-CD3 antibodies with unnatural amino acids, alone or in combination with other suitable components, can also be made into aerosol formulations (i.e., "nebulized") to be administered via inhalation. The aerosol formulations can be placed into pressurized propellants (e.g., dichlorodifluoromethane, propane, nitrogen, etc.).

[0236] Suitable formulations for parenteral administration (e.g., intraarticular (into a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous or non-aqueous isotonic sterile injection solutions. The injection solutions may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the patient's blood, as well as aqueous and non-aqueous sterile suspensions (which may contain suspending agents, solubilizing agents, thickeners, stabilizers, and preservatives). Packaged anti-CD3 antibody formulations may be contained in single-dose or multi-dose containers (such as ampoules and vials).

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

[0238] In the context of the present invention, the dosage administered to a patient is an amount sufficient to induce a long-term beneficial therapeutic response in the patient, or, depending on the application, may be, but is not limited to, an amount that prevents infection by a pathogen or inhibits other suitable activity. The dosage will be determined based on (i) the efficacy of the particular 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. The single dose will also be determined based on the lifestyle and constitution of the particular patient and the extent of any undesirable side effects associated with the administration of the particular vector, formulation, etc.

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

[0240] For example, the dosage administered to a 70 kg patient will typically be in the range equivalent to dosages of currently used therapeutic proteins, adjusted for any altered activity or serum half-life of the composition. The vectors of the invention can complement the treatment of a condition with any known therapy, including administration of antibodies, vaccines, and cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, and the like.

[0241] The formulations of the invention are administered at a rate determined by the LD or ED of the formulation and / or by observing any adverse effects of varying concentrations of the unnatural amino acid, which may be influenced by, but are not limited to, the patient's weight and overall health. The administration may be completed in a single dose or in divided doses.

[0242] If a patient receiving an infusion of the formulation complains of fever, chills, or muscle aches, administer an appropriate amount of aspirin, ibuprofen, acetaminophen, or other antipyretic and analgesic. Patients who experience reactions to the infusion, such as fever, muscle aches, and chills, should be premedicated with, but not limited to, aspirin, acetaminophen, or diphenhydramine 30 minutes prior to further infusion. For more severe chills and muscle aches that do not respond promptly to antipyretics and antihistamines, meperidine should be used. Depending on the severity of the reaction, the pace of cell infusion may be slowed or stopped.

[0243] The human antigen-binding polypeptides of the present invention can be administered directly to a mammalian subject. Administration can be by any route typically used for introducing anti-CD3 antibodies into a subject. Anti-CD3 antibody compositions according to embodiments of the present invention include those suitable for oral, rectal, topical, inhalation (such as, but not limited to, by aerosol), buccal (including, but not limited to, sublingual), vaginal, parenteral (including, but not limited to, subcutaneous, intramuscular, intradermal, intraarticular, intrathoracic, intraperitoneal, intracerebral, intraarterial, or intravenous administration), local (i.e., to skin and mucosal surfaces, including respiratory tract surfaces), and transdermal administration. However, the optimal route in any given case will depend on the nature and severity of the condition being treated. Administration can be local or systemic. The compound formulations may be contained in single-dose or multi-dose quantities in sealed containers (e.g., ampoules and vials). The anti-CD3 antibodies of the present invention may be prepared as a single-dose injectable mixture with a pharmaceutically acceptable carrier (e.g., solution, suspension, or emulsion, but not limited to these). The anti-CD3 antibodies of the present invention may also be administered by continuous infusion (e.g., using a mini-pump such as an osmotic pump), as a single bolus, or as a sustained-release depot.

[0244] Preparations suitable for administration include aqueous or non-aqueous solutions (sterile isotonic solutions), which may contain antioxidants, buffers, bacteriostatic agents, and solutes that maintain the isotonicity of the preparation. Aqueous or non-aqueous sterile suspensions are also suitable for administration, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Solutions and suspensions can be prepared from sterile powders, sterile granules, and tablets of the type described above.

[0245] Pharmaceutical compositions of the invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and the particular method used to administer the composition. Accordingly, suitable formulations of pharmaceutical compositions of the invention (optionally including pharmaceutically acceptable carriers, excipients, or stabilizers) vary widely (see, e.g., Remington's Pharmaceutical Sciences, 17 th ed. 1985].

[0246] Suitable carriers include: buffers (such as phosphate, borate, HEPES, citrate, and other organic acids); antioxidants (such as ascorbic acid); low molecular weight polypeptides (fewer 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 carbohydrates (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); salt-forming counterions (such as sodium); and / or non-ionic surfactants (such as Tween™, Pluronics™, or PEG).

[0247] The anti-CD3 antibodies of the invention, including antibodies linked to a water-soluble polymer such as PEG, can be administered as sustained-release (or partially sustained-release) systems. Sustained-release compositions include, but are not limited to, fabricated articles in semipermeable polymer matrices, such as, but not limited to, films or microcapsules. Sustained-release matrices include biocompatible materials such as 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 (EP 133,988); polylactide (polylactic acid) (U.S. Pat. Nos. 3,773,919 and 58,481); polyglycolide (polymer of glycolic acid); polylactide-co-glycolide anhydride polymer (copolymer of lactic acid and glycolic acid); copolymer of L-glutamic acid and gamma-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 (phenylalanine, tyrosine, isoleucine, etc.); polynucleotides; polyvinylpropylene; polyvinylpyrrolidone; and silicones. The sustained-release composition may further contain a compound encapsulated in liposomes. Liposomes containing the compound are prepared by methods known per se.See DE 3,218,121, [Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692, 1985], [Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034, 1980], EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641, Japanese Patent Application No. 83-118008, U.S. Pat. Nos. 4,485,045, 4,544,545, and EP 102,324 (all cited publications and patent documents are incorporated herein by reference).

[0248] An anti-CD3 antibody encapsulated in a liposome can be prepared, for example, by the methods described in the following documents: DE 3,218,121, [Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692, 1985], [Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034, 1980], EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641, Japanese Patent Application No. 83-118008, U.S. Patent Nos. 4,485,045, 4,544,545, and EP 102,324. The composition and size of liposomes are well known and can be easily determined empirically by those skilled in the art. Some examples of liposomes are described, for example, in the following: [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 cited publications and patent documents are incorporated herein by reference).

[0249] In the context of the present invention, the dosage administered to a patient should be sufficient to induce a beneficial response in the subject over a long period of time. When anti-CD3 antibodies of the present invention are administered parenterally, the total pharmaceutically effective amount per dose is typically 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 subject to therapeutic discretion. The frequency of administration is also subject to therapeutic discretion. The administration may be more or less frequent than commercially available anti-CD3 antibody products approved for use in humans. The bispecific antigen-binding polypeptides of the present invention can generally be administered by any of the administration routes described above.

[0250] Therapeutic Uses of the Anti-CD3 Antibody Bioconjugates of the Invention The anti-CD3 antibody polypeptides of the present invention are useful for treating a wide range of disorders. The anti-CD3 antibody compositions described herein can be used to modulate immune responses. Modulation of immune responses can include stimulating, activating, enhancing, or upregulating the immune response. Modulation of immune responses can include suppressing, inhibiting, preventing, reducing, or downregulating the immune response. The described anti-CD3 antibody-folate compositions can have the advantage of penetrating solid tumors while the natural folate ligand targets, 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 are methods for treating a subject's condition using the anti-CD3 antibody conjugate or pharmaceutical composition of the present invention. In some cancers, the overexpression of specific cell surface receptors allows for selective targeting of cancer cells with small molecules or drugs while minimizing the impact on healthy cells. For example, 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA), which targets the prostate cancer specific membrane antigen (PMSA), can be conjugated to a T cell surface antigen (anti-CD3)-binding antibody to selectively recruit 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 folate receptor (FR) antigen, which is overexpressed on FR+ cancer cell lines.

[0252] The present invention provides methods for treating cancer by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The cancer may be ovarian cancer (including, but not limited to, eptithelial tumors, stromal tumors, and germ cell tumors). The ovarian cancer may include fallopian tube cancer or primary peritoneal carcinoma. The cancer may be characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. The cancer may be treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. In some embodiments, the present invention provides methods for treating genetic diseases, AIDS, or diabetes by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. In some embodiments, the anti-CD3 antibody or treatment may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally comprising a site-specifically incorporated non-naturally encoded amino acid, optionally conjugated to two folate molecules and two PEGylated molecules. In a further embodiment, the anti-CD3 Fab antibody is conjugated to two folate molecules and two PEGylated molecules via the side chains of an unnatural amino acid.

[0253] The present invention provides anti-CD3 antibodies for use in treating diseases or conditions in cells that highly express folate receptors. The anti-CD3 antibodies of the present invention are used to treat cancers, including, but not limited to, ovarian cancer (including, but not limited to, eptithelial tumors, stromal tumors, and germ cell tumors). Ovarian cancer may include fallopian tube cancer or primary peritoneal carcinoma. The cancer may be characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. Cancer may be treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The anti-CD3 antibodies of the present invention are used to treat, but are not limited to, genetic diseases, AIDS, or diabetes. The anti-CD3 antibodies of the present invention may be used in the manufacture of a medicament for treating diseases or conditions in cells that highly express folate receptors. The anti-CD3 antibodies of the present invention can be used in the manufacture of a medicament for treating cancer, including, but not limited to, ovarian cancer (including, but not limited to, eptithelial tumors, stromal tumors, and germ cell tumors). The ovarian cancer can include fallopian tube cancer or primary peritoneal carcinoma. The cancer can be characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. The cancer can be treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The anti-CD3 antibodies of the present invention can be used in the manufacture of a medicament for treating, but not limited to, genetic diseases, AIDS, or diabetes.

[0254] In some embodiments, the condition to be treated is cancer.Cancer includes, but is not limited to, breast cancer, brain cancer, 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 cancer or disease or condition related to any of these cancers.Carcinoma is cancer that develops in epithelial cells, which are cells that cover the surface of the body, produce hormones, and make up 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 cancer, 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, cutaneous or intraocular melanoma, anal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, urethral cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, brain stem glioma, and spinal axis tumor. In some cases, the cancer is a skin cancer such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or solar keratosis (actinic keratosis). In some embodiments, the cancer is any cancer with highly expressed folate receptor alpha or beta. In some embodiments, the condition being 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 with highly expressed folate receptor alpha or beta. The disease or condition can be a pathogenic infection. The biologically active molecule can interact with a cell surface molecule on infected cells. The biologically active molecule can interact with a molecule on a bacterium, virus, or parasite. The pathogenic infection can be caused by one or more pathogens. In some examples, the pathogen is a bacterium, fungus, virus, or protozoan.Exemplary 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, Salmonea, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia. The pathogen may 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, herpes virus, HIV, influenza virus, measles virus, mumps virus, papillomavirus, parainfluenza virus, poliovirus, respiratory syncytial virus, rubella virus, and varicella-zoster virus. Examples of virus-induced diseases or conditions include, but are not limited to, colds, influenza, hepatitis, AIDS, chickenpox, rubella, mumps, measles, warts, and polio. The disease or condition may be an autoimmune disease or an autoimmune-related disease. An autoimmune disease is a malfunction of the body's immune system, which can trigger the body to attack its own tissues.Examples of autoimmune and autoimmune-related diseases include 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's syndrome, Graves' disease, Hashimoto's disease, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, Kawasaki disease, Lambert-Eaton syndrome, and lupus (SLE). , mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus, polyarteritis nodosa, autoimmune polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, psoriasis, psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff man syndrome, Takayasu's 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 alveolitis, amyloidosis, vasculitis, ankylosing spondylitis, avascular osteonecrosis, Graves' disease, Bell's palsy, bursitis, carpal tunnel syndrome, celiac disease, cholangitis, chondromalacia patellar, chronic hepatitis, chronic fatigue syndrome, Cogan's syndrome, congenital hip dysplasia, costochondritis, Crohn's disease, cystic fibrosis, De Quervain's tendonitis, diabetes-related arthritis, diffuse idiopathic osteoarthritis, discoid lupus, Ehlers-Danlos syndrome, familial Mediterranean fever, fasciitis, fibrositis / fibromyalgia, frozen shoulder, ganglion cyst, giant cell arteritis, gout, Graves' disease, HIV-associated rheumatic disease syndrome, hyperparathyroidism-related arthritis, infectious arthritis, inflammatory bowel syndrome / irritable bowel syndrome, juvenile rheumatoid arthritis, Lyme disease, Marfan's syndrome, Miklos syndrome, and the like. These include, but are not limited to, rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, myofascial pain syndrome, osteoarthritis, osteomalacia, osteoporosis and corticosteroid-induced osteoporosis, Paget's disease, relapsing rheumatoid arthritis, Parkinson's disease, Plummer's 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, spondylitis, Still's disease, systemic lupus erythematosus, Takayasu's (pulseless) disease, tendonitis, tennis elbow / golfer's elbow, thyroid-associated arthritis, trigger finger, ulcerative colitis, Wegener's granulomatosis, and Whipple's disease.

[0256] Pharmaceutical compositions containing anti-CD3 antibodies can be formulated to be effective when administered by various means to human patients suffering from disorders that can benefit from the use of anti-CD3 antibody agonists or antagonists. The effects of anti-CD3 antibody agonists or antagonists include, but are not limited to, antiproliferative, anti-inflammatory, and antiviral effects. This effect may be directed at the condition or disease itself, or at a portion of the condition or disease. The average amount of anti-CD3 antibody may vary and should be determined, inter alia, based on the recommendations and prescriptions of a competent physician. The exact amount of anti-CD3 antibody is a matter of preference, given the specific conditions being treated, the condition of the patient being treated, and the other ingredients in the composition. The present invention also provides for the administration of therapeutically effective amounts of other active ingredients, including, but not limited to, anti-cancer chemotherapeutic or immunotherapeutic agents. Those skilled in the art can readily determine dosages based on the treatment using the anti-CD3 antibody.

[0257] [Example] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0258] Example 1 A set of criteria for selecting a suitable site for incorporating a non-naturally encoded amino acid into an anti-CD3 antibody is described. This example demonstrates a method for selecting a suitable site in the antigen-binding polypeptide CD3 for introducing a non-naturally encoded amino acid. The three-dimensional structure of two anti-CD3 antibody molecules, or the secondary, tertiary, or quaternary structure of the anti-CD3 antibody, can be used to select a suitable site.

[0259] The following criteria were used to evaluate each position in the anti-CD3 antibody for introducing a non-naturally encoded amino acid. That is, the residues (a) should not interfere with binding of the anti-CD3 antibody based on structural analysis of the three-dimensional structure, nor should they interfere with the secondary, tertiary, or quaternary structure of the anti-CD3 antibody; (b) should not be affected by scanning mutagenesis using alanine or homologs; (c) should be surface exposed and should have minimal van der Waals or hydrogen bonding interactions with surrounding residues; (d) may be at one or more locations on the exposed surface of the anti-CD3 antibody; (e) may be at one or more locations of the anti-CD3 antibody that are located in proximity to a second anti-CD3 antibody, or other molecule or fragment thereof; (f) should be either deleted or variable in the anti-CD3 antibody variant; (g) may be conservatively changed as a result of substitution with a non-naturally encoded amino acid; (h) may modulate the conformation of the anti-CD3 antibody itself, or of a dimer or multimer comprising one or more anti-CD3 antibodies, by desirably altering the flexibility or rigidity of the overall structure; (i) highly flexible regions (j) may or may not be in the complementarity determining regions (CDRs). Further calculations are performed on the anti-CD3 antibody molecule using the Cx program (Pintar et al. Bioinformatics, 18, pp 980) to assess 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 a non-naturally encoded amino acid.

[0260] Example 2 We describe the expression of an anti-CD3 antibody with a non-naturally encoded amino acid in Escherichia coli. An introduced translation system equipped with an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS) was used to express the anti-CD3 antibody with a non-naturally encoded amino acid. The O-RS preferentially aminoacylates the non-naturally encoded amino acid with the O-tRNA. The translation system then inserts the non-naturally encoded amino acid into the anti-CD3 antibody in response to the encoded codon of choice.

[0261] Site-specific incorporation of non-naturally encoded amino acids into anti-CD3 antibodies can be achieved by transforming E. coli with 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). The modified anti-CD3 antibody is expressed with high fidelity and efficiency by culturing the transformed E. coli at 37°C in medium containing 0.01–100 mM of the specific non-naturally encoded amino acid. Anti-CD3 antibodies containing non-naturally encoded amino acids are produced in E. coli host cells. The product is obtained as a soluble protein in the periplasm. Purification of anti-CD3 antibodies is well known, and can be confirmed by SDS-PAGE, Western blot analysis, or electrospray ionization-ion trap mass spectrometry.

[0262] [Expression / Suppression] Suppression with a non-naturally encoded amino acid (paraacetylphenylalanine (pAF)): Amber mutations in E. coli were suppressed according to standard protocols. Briefly, the following procedure was used to suppress antibody fragments (Fab) in the periplasm of E. coli. First, the 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 overnight incubation, the bacterial culture was diluted 1:100 in a flask containing Luria-Bertani (LB) medium or Superbroth with shaking and grown at 37°C until an OD of approximately 0.8 was reached. Paraacetylphenylalanine (pAF) was added to a final concentration of 4 mM to induce Fab expression while suppressing the amber codon. The culture was then grown overnight at 25°C.

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

[0264] Cells were harvested by centrifugation and resuspended in periplasm release buffer (50 mM NaPO4, 20% sucrose, 1 mM EDTA; pH 8.0) supplemented with 100 μg / ml lysozyme. The cells were then incubated on ice for 30 minutes. After centrifugation, antibody fragments in the supernatant were immobilized on ProBind beads (Invitrogen; Carlsbad, CA) using the His tag of the antibody fragment. After extensive washing with binding buffer, bound fragments were eluted from the beads with 0.5 M imidazole. The purified fragments were dialyzed against 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, E. coli cells in 15 ml of culture medium were harvested by centrifugation. The cells were then resuspended in 1 ml of lysis buffer (B-PER, Pierce Biotechnology; Rockford, IL) supplemented with 10 μg / ml DNase, incubated at 37°C for 30 min, diluted 1× with Protein Loading buffer (Invitrogen; Carlsbad, CA), and analyzed by SDS-PAGE.

[0265] Example 3 Design and Construction of Humanized Anti-CD3 Genes in the pFUSE Vector—The murine monoclonal anti-CD3 antibody SP34 (Harvard BIDMC) was humanized. Based on in silico analysis and design, three variable heavy chain (vH1.0, vH1.1, and vH1.2) and three variable light chain (vL1.0, vL1.1, and vL1.2) genes (Genewiz, South Plainfield, NJ) were synthesized containing the murine CDR sequences plus various murine framework backmutations on a selected human framework scaffold. Table 2 lists the murine framework residues (backmutations) along with Kabat numbering that 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 restored to the human framework sequences are shown in bold.

[0266] [Table 2]

[0267] As shown in Table 2, there were five murine back mutations in the variable light chain (V36, G46, G49, G57, and V58), and four murine back mutations in the variable heavy chain (N30, A49, I77, and V93). The six short synthetic variable genes shown in Table 2 were cloned into Invivogen heavy chain (HC) and light chain (LC) expression vectors (pFUSE-CHIg-HG1 and pFUSE-CLig-hk, respectively) to obtain plasmids expressing the humanized anti-CD3 SP34 antibody. As shown in Figure 1 and Table 2, the variable light chain vL1.0, which had all five murine back mutations converted to human residues, lost binding to any variable heavy chain (vH) combination.

[0268] Example 4 Expression of humanized anti-CD3 antibodies in the HEK293 transient system: The humanized SP34 antibodies described in the above examples were transiently expressed in HEK293 cells by combining a plasmid containing each vH gene with a plasmid containing each vL gene (cotransfection). Protein concentrations in the cell culture medium from each cotransfection experiment were measured and used directly in PBMC-based CD3 binding assays without further purification. Controls used were a chimeric SP34 construct (positive control) and an irrelevant PSMA antibody construct (negative control). Humanized anti-CD3 antibodies without unnatural amino acid incorporation and with or without HC-DKTHT extension were also expressed in HEK293 cells and characterized (Table 3). These antibodies were used to screen for low-affinity Fab variants. 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 Testing for CD3 binding with activated human and sino-PBMCs: To distinguish the generated humanized anti-CD3 antibodies, binding to human CD3 was tested using activated human PBMCs (n=2). PBMC activation and subsequent fluorescence-based CD3 binding assays were performed as previously described (see, e.g., 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. The binding of the remaining six humanized antibodies to both human and sino-CD3 was titrated using activated human and sino-PBMCs, respectively. As shown in Figures 2A-2F, all six humanized anti-CD3 antibodies (engineered from three vH heavy chains with two vL light chain combinations, as shown in Table 3) retained comparable binding to both human and sino-CD3. Six humanized anti-CD3 antibodies were obtained as follows: Fab1, Fab2, and Fab3 wild-type were generated by combining 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-type were generated by combining 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] Because the six humanized antibodies showed no significant differences in binding to both CD3 substrates, Fab1 (the combination of vH1.2 and vL1.2) was selected to further demonstrate this important aspect of the present invention. For example, Fab1 was used for expression in E. coli for further optimization using a proprietary unnatural amino acid (UAA) incorporation technique with a previously described orthogonal amber suppression system (see, e.g., WO2017 / 079272, WO2012 / 166560, and WO2013 / 192360). A list of the murine framework backmutations required to retain binding activity in these six Fabs is shown in Table 4, which provides a list of the murine framework residues (backmutations) retained in the six humanized anti-CD3 Fabs described in Figures 2A-2F, along with Kabat numbering. Amino acid residues restored 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 synthetic Fab genes into E. coli expression vectors: The synthetic Fab genes were cloned into proprietary standard E. coli expression vectors using a 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 strain W3110B60, and single colonies isolated for each plasmid were purified to create glycerol vials. The glycerol vials served as production clones for E. coli fermentation of these Fab molecules. The amino acid sequences of the four heavy chains and three light chains used in engineering these Fabs are shown in Table 5 as SEQ ID NOs: 10-20.

[0274] [Table 5]

[0275] JPEG0007759805000027.jpg63164

[0276] Three light chain (LL157pAF, LK172pAF, and LS205pAF) pAF mutants (SEQ ID NOs: 18-20) and three double pAF mutants ((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 mutants 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) fermenter production. The inoculum is started from a single glycerol vial, thawed, and 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. Prior to use, the fermenter is cleaned and autoclaved. A specific amount of basal medium is added to the fermenter and steam sterilized. Specific amounts of kanamycin sulfate solution, feed medium, and P2000 antifoam are added to the basal medium prior to inoculation. All solutions added to the fermenter after autoclaving are either 0.2 μm filtered or autoclaved before sterile addition.

[0278] The production fermentor was incubated at a target OD of 0.0004 by aseptically transferring the contents of the inoculum. 600 After inoculation, the medium was sampled at appropriate intervals and the OD 600The temperature, pH, and dissolved oxygen are monitored and controlled at specific set points of 37°C, 7.0, and ≥30%, respectively. pH is controlled by the addition of ammonium hydroxide solution or sulfuric acid. Dissolved oxygen is controlled by varying the agitation speed and by increasing the oxygen content in the sparged air / oxygen mixture. Antifoam agents are added during the fermentation process to control foaming.

[0279] Cell density > OD of 25 600 A bolus of feed medium is added when the cell density reaches an OD of >50. 600 Once the ATP concentration reaches 100kJ / L, feed medium is added at a constant flow rate of 0.094 mL / L starting volume / min for 32 hours, until it decreases to 0.052 mL / L starting volume / min and the fermentation is terminated. Immediately after the start of the feed, a specific amount of a non-naturally encoded amino acid (e.g., pAF) solution is aseptically added to allow the non-natural amino acid to be incorporated into the protein amino acid sequence. Simultaneously, 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 phosphate levels in the medium are depleted. Harvesting begins approximately 48 hours after induction.

[0280] Example 7 Purification and conjugation to folate and PEG-folate: 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 performed at 4°C. Cells are lysed in 100 mM acetic acid, 100 mM NaCl, 1 mM EDTA, pH 3.5 in a volume equal to the initial fermentation volume, yielding a post-lysis product at pH 4.1-4.2.

[0281] After lysis, the WCL is centrifuged at 15,900 x g for 30 minutes at 4°C and filtered (0.8 / 0.2 microns) to remove precipitated proteins and cell debris. Anti-CD3 Fab is then captured from the E. coli WCL supernatant using Capto S cation exchange chromatography. Following the Capto S column, Butyl HP hydrophobic interaction chromatography (HIC) is used as a polishing column to isolate intact anti-CD3 Fab from product-related impurities present in the Capto S elution pool. The Butyl HP elution pool containing intact anti-CD3 Fab is then buffer exchanged into 50 mM acetate, 5% trehalose, pH 4.0, concentrated, and prepared 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.

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

[0283] CD3 Fab folate-5KPEG, CD3 Fab folate-10KPEG, CD3 Fab folate-20KPEG, CD3 Fab folate-(5K)2PEG, and CD3 Fab folate-(10K)2PEG compounds were prepared. The desired folate-PEG (5K, 10K, 20K, 5K2, or 10K2PEG) was added to CD3 Fab in a buffer solution (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 a buffer solution of 50 mM histidine, 100 mM NaCl, 5% trehalose, pH 6.0 using a centrifugal filter (c / o 10K) to obtain the desired CD3 Fab-folate-PEGylated composition. The structure, chemistry, and conjugation of the CD3 Fab-folate conjugate are described herein and shown in Figures 12A-12D.

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

[0285] Example 8 In vitro binding and killing assays: Purified humanized anti-CD3 Fabs with folate conjugated at various heavy chain sites (HA114, HS115, HK129, HT160) were first tested for binding to both human and cyno CD3, along with the corresponding non-binding proteins with pAF at these positions, and the WT protein as a control. Table 6 shows the EC binding of modified anti-CD3 Fab1 proteins purified from E. coli cells to activated human and cyno PBMCs. 50 As shown in Table 6, neither pAF nor conjugated folate at different sites significantly interfered with CD3 binding.

[0286] [Table 6]

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

[0288] As shown in Table 7, EC 50 However, in the presence of 50 nM SFA, cytotoxic EC killing was significantly reduced compared to the absence of SFA due to competitive inhibition by free SFA. 50 A decrease of approximately 15 to 60 times was observed. 50 is human EC 50was approximately 10-fold higher for each mutant.

[0289] [Table 7]

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

[0291] [Table 8]

[0292] Anti-CD3 Fab-folate conjugates at all three different sites of conjugation demonstrated efficient killing of all five cell lines with FRα above 10K. However, no killing was observed with any of the conjugates in the A549 cell line, which has an FRα of 5,700. There appears to be a threshold of approximately 10,000 FRα for efficient killing, but killing activity appears to be independent of the number of FRα above the 10K threshold. In vitro killing EC 50 did not change significantly at the folate conjugation site in the antibody, so the HK129 position was selected as the folate conjugation site for further experiments.

[0293] Example 10 This example demonstrates the effect of either single-site folate-PEG conjugation or the addition of a second folate (difolate) on the in vitro binding affinity and cytotoxic activity of various anti-CD3 Fab1-HK129-pAF molecules.

[0294] Effect of folate-PEG conjugates: Table 9 shows the effect on binding, and Table 10 shows the cytotoxicity for conjugates of folate with either 5K or 20K linear PEG molecules using a bifunctional linker as described herein. A slight decrease in binding (1.7-8.4-fold) was observed for both activated human and cytotoxic PBMCs, depending on the 5K or 20K PEG size. However, cytotoxic activity was dramatically reduced with 20K PEG compared to 5K PEG for both human and cytotoxic PBMCs (3.7-5.6-fold vs. 50-58-fold). 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 double folate (bifolate) conjugates: Table 11 shows the effect of second folate conjugation at two light chain sites (LL157 and LK172) in combination with the heavy chain HK129 site on in vitro binding, and Table 12 shows cytotoxicity. As expected, a slight increase in both binding affinity and cytotoxic activity was observed with the bifolate variant over the HK129 single folate molecule. A slight increase in cytotoxicity of the double versus single folate 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 over the LK172 site for future studies.

[0298] [Table 11]

[0299] [Table 12]

[0300] Summary of Binding Affinity and In Vitro Cytotoxicity: Table 13 summarizes the effect of PEGylation and secondary folate conjugation on the binding and cytotoxic activity of various anti-CD3 Fabs. A slight decrease in binding affinity for both CD3 and FRα resulted in a significant decrease in efficacy, correlating with the size of the PEG. Conjugation of two folate molecules increased affinity for FRα, thereby increasing cytotoxic efficacy, despite some decrease in CD3 binding affinity. Despite the decreased affinity for both targets upon PEG conjugation, the in vitro killing EC50s of all CD3-Fab-folate molecules maintained high efficacy, ranging from 37 to 335 pM.

[0301] [Table 13]

[0302] Example 11 Effect of various effector to target (E:T) cell ratios on SKOV-3 cell cytotoxicity by anti-CD3 Fab1 molecules: To test the effect of E:T ratio on SKOV-3 cell cytotoxicity, 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, the E:T ratio correlated with in vitro killing efficacy, as shown in Table 14. At E:T = 1:1, EC 50 A two-fold increase in EC was observed for the difolate molecule, whereas an eight-fold decrease was seen for the 5KPEG-folate molecule. All three molecules had EC values ​​ranging from 1.69 to 175 pM within E:T ratios of 10:1 to 1:10. 50 and remained highly effective.

[0303] [Table 14]

[0304] Example 12 PEGylation of anti-CD3 Fab1 molecules to extend plasma half-life: The pharmacokinetic properties of anti-CD3 Fab1-folate conjugates with and without PEG conjugation were investigated in rats. Male Sprague-Dawley rats (approximately 7 weeks old) were used. On the day of administration, each animal was weighed. Three rats were intravenously injected via the tail vein with 1 mg / kg body weight of unconjugated or conjugated anti-CD3 antibody samples. At different time points after injection, 500 μl of blood was collected from each rat under CO2 anesthesia. The blood samples were stored at room temperature for 1.5 hours, and then serum was separated by centrifugation (4°C, 18,000 × g for 5 minutes). 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 using an anti-CD3 antibody in vitro activity assay.

[0305] As shown in Figure 3, serum concentrations rapidly declined at the same rate for the two anti-CD3 Fab1-folate and the corresponding unconjugated anti-CD3 Fab1, with serum half-lives of less than 1 hour. In contrast, the serum half-lives of the two PEG-conjugated anti-CD3 Fab1s were significantly prolonged to 6.1 and 10.6 hours for 5KPEG and di5KPEG, respectively. Table 15 shows various PK parameters after IV administration of various anti-CD3 Fab1 molecules in rats. The data show that di5KPEG-difolate prolonged T and significantly increased 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 mutants in HEK293 cells.

[0308] Mouse reversion and germline mutations: Mouse framework residues (backmutations) for both the vH and vL sequences obtained during humanization of the anti-CD3 antibody were examined by reverting them one at a time to human germline residues and examining their effect on antigen binding (deconvolution). For the vH sequence, four mouse framework residues (backmutations) 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 (backmutations) 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 effect 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 cotransfection studies with HEK293 cells along with the original vH and vL plasmids described in the previous examples (Round 0 plasmids in Tables 16-17). A total of 35 transient transfections were performed in the Round 1 screening, and cell culture supernatants were directly used to screen for binding as described below and in the previous examples.

[0312] Based on the results of the round 1 screening, three additional vH plasmids (vH2.4-2.6) (Round 2 plasmids in Tables 16-17) were constructed to evaluate the additive effects of individual backmutations. In a similar manner, a new plasmid for vL (vL2.5) was constructed by combining two mouse backmutations. Furthermore, mouse germline mutations found within the vH and vL CDRs were analyzed in this round for conferring reduced binding. To this end, three new vH plasmids (vH3.1-3.3) (Round 2 plasmids in Tables 16-17) were generated by changing the amino acid at Kabat position N35 in HC-CDR1 and Y52c in HC-CDR2. In a similar manner, a vL plasmid (vL3.1) (Round 2 plasmids in Tables 16-17) was constructed by changing the amino acid at Kabat position K53 in LC-CDR2. Round 2 screening experiments were performed with a total of 43 transfections by selectively combining different vL / vH plasmid pairs.

[0313] Screening for human and cyno CD3 binding: HEK293 culture supernatant was used directly to test for binding to human CD3 in the screening stage. In round 1 experiments, 35 clones were initially tested for binding to human CD3 at three different protein concentrations, and 13 were selected for detailed binding assays to both human and cyno CD3. For each clone, an 11-point binding titration curve was generated for both human (Figure 4A) and cyno CD3 (Figure 4B). Based on this data, Fabs 7-10 were selected as low-affinity candidates for further evaluation (Figures 4A-4B). Similarly, 43 clones were screened in round 2 experiments. After two-stage screening as described above for round 1, 11 low-affinity mutants were obtained, four of which are shown in Figures 5A-5B.

[0314] These 15 low-affinity anti-CD3 Fab mutants (Fabs 7-10 from round 1 and Fabs 11-21 from round 2) were then transferred to an E. coli expression system for unnatural amino acid (UAA) incorporation and further testing was performed, as described in the Examples below. Figure 6A shows the Fab-folate complex of the same wild-type Fab shown in Figure 4A. Figure 6B shows a Fab-folate complex representative of the wild-type Fab obtained from round 2 screening. Table 18 lists the low-affinity mutations and their HC, LC, and Fab IDs. As shown, both mouse framework backmutations and germline CDR mutations played important roles in conferring reduced binding to both human and cyno CD3. In the variable light chain (vL), five mouse backmutated residues at Kabat positions V36, G46, G49, G57, and V58 in LC-CDR2 and one mouse germline residue at Kabat position K53 were responsible for the reduced binding. In the variable heavy chain (vH), four mouse reverted residues at Kabat positions N30, A49, I77, and V93 in HC-CDR1 and two mouse germline residues at Kabat positions N35 in HC-CDR1 and Y52c in HC-CDR2 were involved in the reduced binding.

[0315] [Table 18]

[0316] [Table 19]

[0317] JPEG0007759805000042.jpg217164

[0318] SEQ ID NOs: 21-29 represent the amino acid sequences of the heavy chains with the 5-aa C-terminal extension -DKTHT used to screen for low-affinity mutant Fabs. SEQ ID NOs: 30 and 38 represent the amino acid sequences of these humanized heavy chains without the 5-aa heavy chain C-terminal extension. SEQ ID NO: 39 represents the amino acid sequence of the light chain used to screen for 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 variants produced from E. coli cells using the heavy chain HK129 amber mutation.

[0320] Cloning into E. coli expression vectors: Synthetic genes were designed for all low-affinity mutants disclosed in Table 20 (SEQ ID NOS: 40-62) using an STII-LC-spacer-STII-HC expression cassette structure with an amber TAG stop codon inserted at the heavy chain HK129 position and cloned into proprietary E. coli expression vectors as described in the Examples above. 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 the humanized heavy chain HK129pAF mutants with the 5-aa heavy chain C-terminal extension -DKTHT used in testing. SEQ ID NOS: 49 and 57 represent these humanized heavy chain HK129pAF mutants without the 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, which were expressed in E. coli and further characterized.

[0321] [Table 20]

[0322] JPEG0007759805000044.jpg217164

[0323] JPEG0007759805000045.jpg125164

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

[0325] Binding of folate-conjugated Fabs to human and cytoplasmic CD3: Binding of folate-conjugated low-affinity variants of the E. coli-produced and humanized anti-CD3 Fabs was performed as described in the previous examples. Figures 6A-6B show the binding affinity to human CD3 for two subsets of low-affinity Fab variants of anti-CD3 Fab-HK129-folate, along with the positive control Fab1. Of the low-affinity Fabs tested, 10 failed to significantly bind to human CD3, even at the highest concentration tested (1000 nM). The binding EC values ​​of the remaining four Fabs (Fabs 7-10; listed in Table 18) were 0.01 and 0.02, respectively. 50 The binding activity varied from 23.4 nM to 83.6 nM, compared to 2.31 nM for the control Fab1. Fab21 exhibited weak, non-saturating binding activity even at 1000 nM. A very similar binding profile was observed for cynoCD3 binding, as shown in Figures 7A-7B.

[0326] Cytotoxicity assay of folate-conjugated Fabs using human and sino-PBMCs: Cytotoxicity assays of low-affinity Fabs were performed as described in the previous examples. Figure 8 shows the cytotoxicity data for activated human PBMCs with SKOV-3 cells. As shown, all four Fabs (Figures 6A-6B), which had 10- to 36-fold reduced binding affinity for human CD3, exhibited comparable killing activity compared to the control Fab1 (Figure 8). Three Fabs (Fabs 11, 19, and 20) did not exhibit any cytotoxic activity (data not shown). All eight other Fabs failed to bind even at 1000 nM concentrations but still exhibited significant killing activity. A very similar cytotoxicity profile was observed in activated sino-PBMCs (Figure 9).

[0327] Example 15 This example demonstrates 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 equal volumes of whole blood using the EasySep Human T Cell Enrichment Kit and 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 T cell activation in the presence of accessory cells, 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 anti-CD3 Fab variants. After 48 hours, cells were labeled with APC-Cy7-conjugated anti-human CD25 (Biolegend) or PE-conjugated anti-human CD69 (BD Biosciences) and analyzed by flow cytometry. IFNγ and TNFα release in the culture supernatants was measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D System).

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

[0330] Summary of low-affinity variant characterization: Table 21 shows binding and cytotoxicity data (human and cytotoxic CD3), as well as T-cell activation (CD25 and CD69 markers) and cytokine release (IFNγ and TNFα) analyses for 12 low-affinity anti-CD3 Fab variants with HK129-folate modifications. As shown in the figure, there is a general correlation between CD3 binding strength, cytotoxicity, T-cell activation, and cytokine release.

[0331] [Table 21]

[0332] Based on the data set, three low affinity mutants, Fab9, Fab10, and Fab21, were selected along with the parent molecule Fab1 for detailed in vitro characterization, including in vivo studies in mice. Table 22 shows a comparison of these mutants with respect to cytotoxicity and production of two cytokines. As shown, cytokine production is associated with increased EC2 activity for T cell activation and killing. 50 Much higher EC than 50 Therefore, it may be possible to identify an anti-CD3 Fab concentration range at which cytokine release does not pose a significant safety issue, but efficacy and T cell activation killing are not compromised. This approach to fine-tuning anti-CD3 antibody affinity allows for the separation of these two opposing events, which may achieve a better safety profile without compromising efficacy.

[0333] [Table 22]

[0334] Example 16 In silico immunogenicity analysis of anti-CD3 Fabs 1, 9, and 10: To assess potential immunogenicity, the amino acid sequences of anti-CD3 Fabs 1, 9, and 10 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" configuration (Lonza, UK) based on Lonza's Epibase platform. The HLA-binding specificity of all possible 10-mer peptides derived from the target sequences was analyzed. Profiling was performed at the allotype level for 43 DRB1, 8 DRB3 / 4 / 5, 22 DQ, and 12 DP HLA class II allotypes, totaling 85 allotypes. Peptides corresponding to self-peptides were treated separately as "germline-filtered" peptides. As a general summary of the results, Table 23 shows the number of strong binders corresponding to the DRB1, DRB3 / 4 / 5, DQ, and DP genes (epitope number). As with humoral responses generated against antigens, the observed Th cell activation / proliferation is generally interpreted in terms of DRB1 specificity. The results in Table 23 show that Fab 1, Fab 9, and Fab 10 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 worldwide populations comparable to those of humanized treated antibodies. Of the three Fabs, Fab 9 is the least immunogenic.

[0335] [Table 23]

[0336] [Table 24]

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

[0338] [ka]

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

[0340] [ka]

[0341] N 10 N-trifluoroacetylpteroic acid (2). 1.0 g of pteroic acid (1) was added dropwise to 10 ml of trifluoroacetic anhydride in a round-bottom flask under nitrogen for 10 minutes. The reaction mixture was stirred at room temperature for 24 hours, away from light. 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 under vacuum overnight to give 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, and the precipitate was collected by filtration, washed with ether, and dried under vacuum overnight to give N-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). N in anhydrous DMSO 10 A solution of 2-trifluoroacetylpteroic acid was treated with N-hydroxysuccinimide (0.43 g) followed by EDCI-HCl (2.04 g) in one portion at room temperature. 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, and dried under vacuum for 1 day to give compound 3 as a brown powder, MS(ESI) m / z 506 (M+H). + was obtained as.

[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) was added DCC (2.06 g) in one portion at room temperature. The resulting solution was stirred overnight at room temperature, and 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) was obtained as a white foam. This material was redissolved in THF (20 mL) and added to a mixture of H-Lys(Boc)-OtBu-HCl (6) (3.39 g) and DIPEA (3.5 mL) in anhydrous THF (50 mL) at room temperature. The resulting mixture was stirred at ambient temperature for 4 hours until the reaction was judged complete by HPLC analysis, and the solvent was removed under vacuum. The residue was redissolved in ethyl acetate (100 mL), washed with 10% aqueous citric acid (50 mL), water (50 mL), and brine (50 mL), and dried over sodium sulfate. After the solvent was removed under vacuum, the residue was triturated with 5% ether / hexane (50 ml). The isolated 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) was obtained.

[0344] H-Glu-O t Bu-Lys(Boc)-O t Bu(8). H-Glu-O in DCM t Bu-Lys(Boc)-O t The solution of Bu(7) was treated with diethylamine. The resulting solution was stirred at room temperature for 4 hours until deprotection was complete as judged 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) was obtained as a colorless oil.

[0345] Compound 9: A solution of amine 8 in anhydrous DMF was treated with N 10 The resulting mixture was treated with 1-trifluoroacetylpteroic acid OSu ester (3) in one portion. The resulting mixture was stirred at room temperature for 24 hours while being monitored for completion 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 allowed to stand at room temperature for 2 hours until total deprotection was complete, as determined by HPLC analysis. All solvents were removed under vacuum, and the residual brown oil was triturated with diethyl ether and briefly sonicated. 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 a synthetic route for the synthesis of compound 13.

[0348] [ka]

[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 overnight at room temperature 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 the eluent to give compound 12 as a white solid after lyophilization.

[0350] Compound 13: To a solution of compound 12 (0.31 g) in DMF (1.5 mL) was added hydrazine, H2O (0.19 mL) at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (approximately 2 mL) and purified by C18 reverse-phase HPLC using a 20-90% acetonitrile / 0.05% TFA gradient as the eluent to give compound 13 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 for the synthesis of compound 11 (CAS#: 1415328-95-8).

[0352] [ka]

[0353] Compound 16: To a solution of tetraethylene glycol 14 in anhydrous THF, small pieces of sodium were added at room temperature and stirred until completely dissolved. To the resulting solution, acrylate 15 was slowly added over 15 minutes. The reaction mixture was stirred at room temperature for 20 hours, then concentrated in vacuo, 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 afforded compound 16 as a clear, yellowish oil.

[0354] Compound 17: To a mixture of alcohol 16 and pyridine in anhydrous DCM was added tosyl chloride in small portions 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 removing the solvent, the residue was purified on 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 deep 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 on 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 h, the solvent was removed in vacuo, and the residue was taken up in dichloromethane, washed thoroughly with brine, and dried over sodium sulfate. After removing the solvent in vacuo, the crude carboxylic acid 19 was obtained as a clear, yellowish oil.

[0357] Compound 11: The crude carboxylic acid 19 in anhydrous THF was treated with N-hydroxysuccinimide, followed by DCC at room temperature. Stirring was continued for 6 h, and 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 branched linker synthesis. For example, the synthetic route for the synthesis of compound 25:

[0359] [ka]

[0360] Compound 21: Boc-Lys-OH 20. To a solution of OSu ester 11 (1.55 g) and Boc-Lys-OH 20 (0.72 g) in DCM (50 ml) was added DIEA (1.03 mL) at 23 °C. After 10 min, LCMS showed the reaction was complete. The mixture was washed with 1 N HCl (50 ml), saturated sodium bicarbonate (50 ml), and brine (50 ml). The organic layer was dried over MgSO. Removal of the solvent gave crude acid 21 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 and then filtered to remove DCC and washed with THF. The product was isolated by silica gel column chromatography using a 0-10% methanol / DCM gradient as eluent to give compound 22 as a white solid (MS(ESI) m / z 737 (M+H)). + ) was obtained.

[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, 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 DCC and concentrated in vacuo. The residue was purified by silica gel column chromatography using a 0-6% methanol / DCM gradient as eluent to give compound 25 as a white solid (MS(ESI) m / z 984 (M+H)). + ) was obtained.

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

[0365] [ka]

[0366] This example describes the synthesis of branched PEG-folate compound 30.

[0367] [ka]

[0368] [ka]

[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 h. The mixture was purified by Prep-LC using a C18 column with a 5% to 60% water / 90% ACN 0.05% TFA gradient over 20 min. The product-containing fractions were combined and evaporated in vacuo to give compound 26 as a brown solid; MS (ESI) m / z 1535 (M+H). + .

[0370] Compound 27: Compound 26 (0.25 g) was added with DCM (3 ml) and TFA (2 ml) at 23° C. and then stirred for 30 minutes. The solvent was removed in vacuo. The residue was dissolved in DCM (approximately 5 ml) and added dropwise to 45 ml of MTBE in a conical tube. The precipitate was isolated by centrifugation (4000 rpm, 5 min) 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) was added DIEA (0.034 mL) at 23 °C. After stirring for 5 h, the mixture was added dropwise to 40 mL of MTBE and centrifuged (5 min, 4000 rpm) to separate the precipitate. To the precipitate, 45 mL of MTBE was added and centrifuged (5 min, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to give compound 29A as a crude white solid.

[0372] Compound 30A: To a solution of compound 29A (0.24 g) in water (10 ml), hydrazine, HO (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 20% to 100% ACN and 0.05% TFA water gradient over 20 minutes. The product-containing fractions were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to give 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) was added DIEA (0.040 mL) at 23 °C. After stirring for 16 h, the mixture was added dropwise to 40 mL of MTBE and centrifuged (5 min, 4000 rpm) to separate the precipitate. To the precipitate, 45 mL of MTBE was added, followed by centrifugation (5 min, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to give compound 29B as a crude white solid.

[0374] Compound 30B: To a solution of compound 29B (0.47 g) in water (10 ml) was added hydrazine, HO (0.080 ml) at 23 °C. After stirring for 6 h, the mixture was purified by Prep-LC using a C18 column with a 20% to 100% ACN and 0.05% TFA water gradient over 20 min. The product-containing fractions were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to give 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 added dropwise to 40 mL of MTBE and centrifuged (5 min, 4000 rpm) to separate the precipitate. To the precipitate, 45 mL of MTBE was added and centrifuged again (5 min, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to give 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, HO (0.060 ml) at 23 °C. After stirring for 6 h, the mixture was purified by Prep-LC using a C18 column with a 20% to 100% ACN and 0.05% TFA water gradient over 20 min. The product-containing fractions were combined and evaporated. The residue was dissolved in water (10 ml) and lyophilized to give 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 added dropwise to 40 mL of MTBE and centrifuged (5 min, 4000 rpm) to separate the precipitate. To the precipitate, 45 mL of MTBE was added and centrifuged (5 min, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to give compound 29D as a crude white solid.

[0378] Compound 30D: To a solution of compound 29D (0.45 g, <0.021 mmol) in water (8 ml) was added hydrazine, HO (0.080 ml) at 23 °C. After stirring for 48 h, the mixture was purified by Prep-LC using a C18 column with a 20% to 100% ACN and 0.05% TFA water gradient over 20 min. The product-containing fractions 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: To a solution of compound 28E1 ((PEG5K)2-NHS, 0.1 g) and aminovaleric acid (0.003 g) in DMF (0.5 mL) was added DIEA (0.010 mL) at 23 °C. After stirring for 1 hour, the mixture was diluted with water to 1 mL and purified on a desalting column. The collected fractions were lyophilized to give compound 28E as a white solid.

[0380] Compound 30E: To a solution of compound 28E (0.04 g), DMTMMT (0.003 g), and DIEA (0.005 mL) in DMF (2 mL) was added compound 27 (0.009 g) at 23 °C. After stirring for 1 h, LCMS showed the reaction was complete. To this mixture (crude compound 29E) was added hydrazine, and HO (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% to 100% ACN and 0.05% TFA water gradient over 20 min. The product-containing fractions were combined and evaporated. The residue was dissolved in water (10 mL) and lyophilized to give compound 30E as a pale yellow solid.

[0381] [ka]

[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) was added DIEA (0.006 mL) at 23 °C. After 30 minutes, the mixture was purified on a desalting column (PD-10) and lyophilized overnight to give 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 double amber-containing humanized anti-CD3 Fab lead molecules. 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 the new Fab molecules Fab1-HK129pAF-LL157pAF, Fab9-HK129pAF-LL157pAF, and Fab10-HK129pAF-LL157pAF, respectively. This allowed for the conjugation of two folate and two 5KPEG molecules in each Fab using either bifunctional linker, as described in the previous example.

[0384] The purified CD3-PEG-folate protein used for in vitro activity, in vivo efficacy, and PK studies was analyzed by SDS gel electrophoresis (SDS-PAGE). Purified CD3 was conjugated to 5K or 10K PEG-folate at the pAF site using oxime chemistry, then purified. Post-conjugation, cation exchange chromatography was used to separate unconjugated, single-site, and dual-site conjugated forms (Figures 13A-13B). After purification, the composition was formulated 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 conjugated with 5K PEG. Lanes 3 and 6 represent unconjugated 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-BiFolate, respectively.

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

[0387] To increase conjugation efficiency, purified CD3Fab compositions were conjugated with PEG-folate using a two-step conjugation method. Folate was conjugated at the pAF site using oxime chemistry, followed by PEG conjugation to 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 analysis under non-reducing (lanes 2-5) and reducing (lanes 7-10) conditions with 10 μg of protein loaded per well. Lanes 2 and 7 represent the Bi-folate unconjugated composition, while lanes 3 and 8, 4 and 9, and 5 and 10 represent the Bi-folate-C-terminal Bi5KPEG, Bi10KPEG, and Bi20KPEG compositions, respectively. The data in Figure 13C demonstrate high purity (>95%) for all samples, demonstrating the expected increase in molecular weight based on the size of the PEG.

[0388] Example 19 This example demonstrates the effect of Bi-Folate and Bi-PEG conjugation on 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-BiFolate conjugation, on the binding affinity and cytotoxicity of Fab1 molecules in the presence of 50 nm SFA.

[0389] [Table 25]

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

[0391] [Table 26]

[0392] Table 27 compares the binding affinity of 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-BiFolate using the PEG-folate bifunctional linker resulted in reduced human CD3 binding and corresponding cytotoxic potential for all three Fabs compared to their unconjugated controls, as described in the Examples above.

[0393] [Table 27]

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

[0395] Example 20 In vitro cytotoxicity data show that CD3-folate bispecific antibodies selectively kill FOLRα-expressing KB cells. KB cells were treated with increasing concentrations of CD3-folate bispecific antibodies in the presence of 50 nM folate (a physiologically relevant concentration of folate). The most potent CD3-folate bispecific antibody, Fab1-HK129-LL157-Bifolate, contains two molecules of folate and has an IC of 1.3 pM. 50 The results showed that the CD3-folate bispecific antibody containing a single folate, Fab1-HK129-folate, exhibited an IC50 of 40.3 pM, 31-fold less potent than Fab1-HK129-LL157-Bifolate. This data indicates that the addition of two folates increases the efficacy of the CD3-folate bispecific antibody. The addition of 5KPEG reduced 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, with an IC 50 The IC value was 198 pM. A 37.3-fold decrease in efficacy was observed between Fab1-HK129-LL157-BiFolate and Fab1-HK129-LL157-Bi5KPEG-BiFolate, with an IC 50The value was 48.5 pM. These data demonstrated that the CD3-folate bispecific antibody retains potent in vitro cytotoxicity at physiologically relevant concentrations of folate.

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

[0397] [Table 28]

[0398] Example 22 Further in vitro cytotoxicity studies were performed in the presence of 20 or 50 nM 5-mTHF. SKOV3 cells were treated with increasing concentrations of CD3-folate bispecific antibodies in the presence of 20 or 50 nM 5-methyltetrahydrofolate (5-mTHF), a physiologically relevant concentration of the major form of folate found in human serum (Figures 16A and 16B). 5-mTHF has a binding affinity of 1 to 10 nM for FOLRα and does not bind to FOLRα as well as folate, which has a binding affinity of less than 1 nM. The CD3-folate bispecific antibodies maintained highly potent IC50 values ​​of 0.03 to 0.16 pM and 0.1 to 1.5 pM for the dual and single CD3-folate bispecific antibodies, respectively. The data in Table 29 demonstrate that the CD3-folate bispecific antibodies have 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 pharmacokinetic study in CD1 mice: CD3-folate bispecific antibody was administered intravenously via the mouse tail vein of CD1 mice at 1 or 5 mg / kg. Blood samples were collected at nine time points and analyzed by ELISA. Data clearly showed that the addition of 5KPEG increased serum exposure (AUClast) (Tables 30-31 and Figure 17). Fab1-HK129-5KPEG-folate at 1 mg / kg showed a 4.3-fold improvement over Fab1-HK129-folate, and Fab1-HK129-5KPEG-folate at 5 mg / kg showed a 5-fold improvement over Fab1-HK129-folate. Fab1-HK129-LL157-Bi5KPEG-Bifolate showed a 16.25-fold improvement over Fab1-HK129-LL157-Bifolate at 1 mg / kg, and Fab1-HK129-LL157-Bi5KPEG-Bifolate showed a 21.7-fold improvement over Fab1-HK129-LL157-Bifolate at 5 mg / kg. The data show a 3.9-fold difference between Fab1-HK129-5KPEG-folate and Fab1-HK129-LL157-Bi5KPEG-Bifolate. Improvements in serum half-life (T1 / 2) were also observed when 5KPEG was added to the CD3-folate bispecific antibody. The greatest improvement in serum half-life was observed with two 5KPEGs incorporated into the CD3-folate bispecific antibody. Fab1-HK129-LL157-Bi5KPEG-Bifolate had a 4.2-fold improved serum half-life over Fab1-HK129-LL157-Bifolate at 1 mg / kg, and Fab1-HK129-LL157-Bi5KPEG-Bifolate had a 6.25-fold improved serum half-life over Fab1-HK129-LL157-Bifolate at 5 mg / kg. The data indicate that the addition of 5KPEG improves serum exposure and serum half-life of CD3-folate bispecific antibodies, reducing the dosing frequency required to achieve effective serum exposure in vivo.

[0401] [Table 30]

[0402] [Table 31]

[0403] Example 24 This example demonstrates that CD3-folate-folate bispecific antibodies kill human M2 macrophages. Macrophages are a heterogeneous cell population that play a role in host defense. While classically activated macrophages (M1 macrophages) have proinflammatory functions, tumor-infiltrating lymphocyte recruitment, and antitumor activity, 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 may enhance the activity of immuno-oncology treatments such as checkpoint inhibitors. Human M2 macrophages express FOLRβ or FRβ, which have similar binding affinity for folate as FOLRα or FRα. Therefore, strategies to increase the ratio of M1 to M2 macrophages by repolarizing them to M1 macrophages or selectively killing M2 macrophages offer potential therapeutic approaches for cancer treatment.

[0404] Test 1: To evaluate the effect of CD3-folate bispecific antibodies 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. Data showed that Fab1-HK129-LL157-folate and Fab1-HK129-LL157-5KPEG-folate had IC values ​​of 1.2 pM and 112.1 pM, respectively. 50These results demonstrate that CD3-folate bispecific antibodies kill human M2 macrophages at 500 ng / mL (data not shown). Fab1-HK129-L157-pAF, lacking folate, does not kill human M2 macrophages. These data confirm that the cytotoxicity of Fab1-HK129-LL157-folate and Fab1-HK129-LL157-5KPEG-folate is specific to binding to FOLRβ. Furthermore, these results suggest that in addition to killing FOLRα-expressing tumor cells, the CD3-folate bispecific antibody may also kill M2 macrophage cells and possibly other FOLRα / β-expressing immunosuppressive cells.

[0405] Study 2: In these studies, 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] Prior to conducting the study, folate receptor β (FOLRβ or FR-β) expression was first measured by flow cytometry and found to be increased by an average of 26-fold in M2 macrophages compared to M1 macrophages in terms of median fluorescence intensity (MFI).

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

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

[0409] [Table 32]

[0410] The results of in vitro macrophage cytotoxicity by CD3-folate bispecific antibodies containing a single folate from a representative donor, donor 6007, are shown in Figure 18A. Arrows and numbers indicate the fold difference between M1 and M2 macrophages in 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 for in vitro macrophage cytotoxicity by dual-folate CD3-folate bispecific antibodies in the presence of 20 nM folate. 50 Data shown: Fab1-HK129-LL157-BiFolate had an average IC of 29.2 pM in M1 macrophages. 50 (range 10.9–42.8 pM), with a mean IC of 1.5 pM in M2 macrophages 50 (range 0.1 pM–5.5 pM) with a mean IC between M1 and M2 macrophages 50 The ratio difference was 72-fold (range 6-212-fold). Fab1-HK129-LL157-BiFolate-Bi5KPEG had an average IC of 1620.8 pM in M1 macrophages. 50 (range 834.7–2651 pM), with an average IC of 15.7 pM in M2 macrophages 50 (range 3.6–52.4 pM), showing the mean IC between M1 and M2 macrophages. 50 The ratio improved to 181-fold (range 49-501-fold) for M2 macrophages. These results indicate that the CD3-folate bispecific antibody with double folate is specific for killing M2 macrophages. PEGylation of the CD3-folate bispecific antibody with double folate (Fab1-HK129-LL157-Bifolate-Bi5KPEG) was shown to be less effective than Fab1-HK129-LL157-Bifolate or Fab1-HK129-5KPEG-Folate, as shown in Table XXX, but was more selective for killing M2 macrophages.

[0412] [Table 33]

[0413] Figure 18B shows the results of in vitro macrophage cytotoxicity by CD3-folate bispecific antibodies containing dual folates from a representative donor, donor 6007. Arrows and numbers indicate the fold difference (IC) between M1 and M2 macrophages. 50The 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.

[0414] Further studies were conducted to mimic the physiologically relevant concentrations of the major forms of folate found in human serum, which range from 9.1 to 45.1 nM. Of this physiological range, 86.7% (37.5 nM) is the primary folate metabolite, 5-methyl-tetrahydrofolate (5-mTHF), and only 4% (1.2 nM) is unmetabolized folate (Pfeiffer et al., Br. J. Nutr., 2015 June 28:113(12):1965-1977). As is known in the art, the binding affinity of folate to FR-β is less than 1 nM, and the affinity of 5-mTHF to FR-β is 1 to 10 nM. Therefore, it was hypothesized that the concentration or composition of folate and 5-mTHF may affect the activity of CD3-folate bispecific antibodies. To assess this, the following experiment was performed: In vitro macrophage cytotoxicity data in the presence of 45 nM 5-mTHF are shown in Tables 34 and 35. IC of Fab1-HK129-Folate 50 The mean IC values ​​for Fab1-HK129-5KPEG-folate were 9.1 pM and 0.31 pM in M1 and M2 macrophages, respectively. 50 The mean IC of Fab1-HK129-LL157-Bifolate was 48.5 pM and 1.26 pM in M1 and M2 macrophages, respectively. 50 The mean IC of Fab1-HK129-LL157-Bifolate-Bi5KPEG was 4.0 pM for M1 and 0.05 pM for M2. 50 The IC between M1 and M2 was 99.4 pM and 0.85 pM, respectively. These data indicate that the CD3-folate bispecific antibody is more potent in the presence of 5-mTHF than folic acid (Tables 32 and 33). 50The ratios were an average of 43-fold and 57-fold for the CD3-folate bispecific antibody with a single folate, and an average of 89-fold and 212-fold for the CD3-folate bispecific antibody with a double folate. These results indicate that the CD3-folate bispecific antibody maintains M2 macrophage-specific killing, and that PEGylation confers greater 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 doubly PEGylated CD3-folate composition exhibited greater specificity for killing M2 macrophages than the single PEGylated CD3-folate composition, and thus improved selectivity was observed with greater PEGylation.

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

[0419] Based on the observations, the proportion of mMDSCs decreased in a dose-dependent manner. Fab1-HK129-5KPEG-Folate showed a dose-dependent reduction from 72% to 24% at 10 pM vs. 100 pM, respectively. Similarly, Fab1-HK129-LL157-Bi5KPEG-BiFolate showed a dose-dependent reduction from 88% to 22% at 10 pM vs. 100 pM, respectively. Given the preservation of non-MDSCs with high HLA-DR activity, treatment with CD3-Folate bispecific antibodies was shown to selectively eliminate mMDSCs.

[0420] Assuming that these suppressor cells may express folate receptors and are involved in suppressing the activity of immuno-oncology drugs, it is suggested that combination therapy with checkpoint inhibitors and other immuno-oncology drugs may be useful as a treatment for various cancers or conditions / diseases / disorders in which folate receptors may be expressed, supporting the use of the CD3-folate composition of the present invention as a valuable therapeutic agent that is distinct from other immuno-oncology therapies.

[0421] Example 25 This example demonstrates the binding affinity of CD3-folate bispecific antibodies to FOLRα in several cell types. CD3-folate bispecific antibody binding affinity to FOLRα-expressing KB cells: As shown in Table 36, Fab1-HK129-folate binds to FOLRα-expressing KB cells with an affinity of 1.97 nM, and Fab1-HK129-5KPEG-folate binds to KB cells with an affinity of 3.69 nM. Fab1-HK129-LL157-Bifolate binds to KB cells with an affinity of 0.85 nM, and Fab1-HK129-LL157-Bi5KPEG-Bifolate binds to KB cells with an affinity of 2.28 nM. The PEGylated antibodies retained similar binding affinity to the non-PEGylated antibodies in the low nM range of FOLRα-expressing KB cells.

[0422] [Table 36]

[0423] CD3-folate bispecific antibody binding affinity to human T cells: Fab1-HK129-pAF (anti-CD3 Fab) binds to human T cells with an affinity of 1.59 nM without folate or PEG (Table 37). Fab1-HK129-folate binds to CD3-expressing human T cells with an affinity of 1.53 nM, and Fab1-HK129-5KPEG-folate binds to human T cells with an affinity of 2.34 nM. These CD3-folate bispecific antibodies have similar binding affinities to human T cells. Fab1-HK129-LL157-pAF binds to human T cells with an affinity of 0.604 nM. Fab1-HK129-LL157-BiFolate binds to human T cells with an affinity of 0.669 nM, and Fab1-HK129-LL157-Bi5KPEG-BiFolate has an affinity of 3.34 nM. These data show that the pegylated CD3-folate bispecific antibody has approximately 5-fold lower binding affinity to human T cells than the non-pegylated version. These data also show that the CD3-folate bispecific antibody has low nM binding affinity to human T cells.

[0424] [Table 37]

[0425] CD3-folate bispecific antibody binding affinity to cynomolgus monkey T cells: As shown in Table 38, Fab1-HK129-pAF binds to cynomolgus monkey T cells with an affinity of 2.67 nM. Fab1-HK129-folate binds to CD3 expressing cynomolgus monkey T cells with an affinity of 2.69 nM, and Fab1-HK129-5KPEG-folate binds with an affinity of 3.31 nM. This indicates that CD3-folate bispecific antibodies with a single amber site have similar binding affinities to cynomolgus monkey T cells. CD3-folate constructs with dual amber sites showed similar binding affinities in cynomolgus monkey T cells. As shown in Table 38, Fab1-HK129-LL157-pAF binds to cynomolgus monkey T cells with an affinity of 0.995 nM. Fab1-HK129-LL157-BiFolate binds to cynomolgus T cells with an affinity of 1.0 nM, and Fab1-HK129-LL157-Bi5KPEG-BiFolate binds with an affinity of 5.01 nM. Thus, the data further indicate that the PEGylated CD3-folate bispecific antibody has approximately 5-fold lower binding affinity for cynomolgus T cells than the non-PEGylated antibody. Overall, the data indicate that the CD3-folate bispecific antibody has low nM binding affinity for cynomolgus T cells.

[0426] [Table 38]

[0427] Example 26 The following study demonstrates an in vivo safety and efficacy study in mice using the CD3-folate antibodies of the invention.

[0428] Study 1: Anti-CD3-folate bispecific antibodies were tested for anti-tumor efficacy by repeated administration to female immunodeficient mice bearing human cervical tumors derived from the KB cell line. A non-targeting anti-CD3 antibody was included as a control. Female NSG mice bearing KB cervical tumors (2.0 x 10 cells from passage 7) were treated with 2.0 x 10 6 The cells were inoculated and initially expanded from the third frozen passage) and divided into five groups of eight animals each.3 In the case of , 7.5 × 10 6 Mice were inoculated intraperitoneally with PanT cells. 24 hours later, mice were randomized into the following treatment groups: G1: anti-CD3Fab1-HK129-pAF control (0.05 mpk), G2 and G3: CD3Fab1-HK129-L157-Bifolate-Bi5KPEG at 0.01 mpk and 0.05 mpk, respectively, G4: Fab9-HK129-L157-Bifolate-Bi5KPEG (0.125 mpk), and G5: CD3Fab1-HK129-Folate control (0.25 mpk); tumor volume (Figure 19A) and mass / body weight (Figure 19B) were measured. All mice were intravenously (IV) dosed on day 7, at which time tumors averaged approximately 125 mm. 3 Animals were monitored twice weekly for tumor growth by caliper measurements and body weight. All CD3-Folate bispecific compositions were effective in Group 3 (Fab1-HK129-LL157-BiFolate-Bi5KPEG, 0.05 mpk), which had the greatest effect on tumor growth inhibition (TGI=82%), as shown in Figure 19A. All compositions, except the control CD3Fab-HK129-pAF antibody, caused weight loss (Figure 19B).

[0429] Biomarker Analysis: Blood samples were collected from each animal before treatment initiation, and on days 7 and 24 after treatment initiation. Samples were analyzed for the ratio of human CD45 / mouse CD45 by FAC analysis to determine whether treatment increased peripheral human T cell populations, as indicated by CD3-folate activation / targeting. At the end of the study, tumors were analyzed by FAC for the presence of tumor-infiltrating lymphocytes (TILs) using the human lymphocyte marker hCD45. All CD3-folate treatment groups demonstrated antitumor efficacy, increasing blood levels of human CD45 to varying degrees, with the highest levels of human CD45 achieved by treatment with the bispecific antibody Fab1-HK129-LL157-Bifolate-Bi5KPEG (0.05 mpk) (Figure 19C). This study also examined the ability of the inventive CD3-folate composition to inhibit tumor growth and promote TILs. Furthermore, the presence of TILs, a hallmark of activated tumor immune surveillance, was increased in all treatment groups compared to the control group, with Group 3 (Fab1-HK129-LL157-BiFolate-Bi5KPEG, 0.05mpk) being found to have the highest induction of TILs (Figure 19D).

[0430] Study 2: This study investigated tumor growth inhibition (TGI) in NCG mice bearing KB tumors and changes in the ratio of human CD45 / tumor infiltrate in mouse blood. Single (Fab1-HK129-LL157-Folate-5KPEG) and double (Fab1-HK129-LL157-BiFolate-Bi5KPEG) PEGylated anti-CD3-folate bispecific antibodies were tested for anti-tumor efficacy by repeated administration to female immunodeficient mice bearing human cervical tumors derived from the KB cell line. A non-targeting CD3 antibody was included as a control. Thirty-five female NCG mice bearing KB cervical tumors (2.0 × 10 cells from passage 5) were injected with PEGylated anti-CD3-folate bispecific antibodies. 6 The cells were inoculated and initially propagated from the third frozen passage) and divided into three groups of 10 animals each. 3 If the mouse is 6.0x10 6PanT cells were inoculated intraperitoneally. 24 hours later, mice were randomized into the following treatment groups: G1: CD3Fab1-HK129-pAF control, G2: Fab1-HK129-LL157-Folate-5KPEG, G3: Fab1-HK129-LL157-BiFolate-Bi5KPEG, each at 0.025 mpk every 5 days (Figures 20A and 20B). All mice received 6.0 × 10 6 Pan-T cells were inoculated and administered intravenously (IV) on day 8, resulting in tumors averaging approximately 100 mm 3 Animals were monitored twice weekly for tumor growth (Figure 20A) by caliper measurement and body weight (Figure 20B). All CD3-folate-targeted compositions showed varying degrees of efficacy, with G2:Fab1-HK129-LL157-Folate-5KPEG (single PEG composition) having the greatest impact on tumor growth inhibition (TGI = 85%), causing complete tumor regression (CR) in 5 of 10 mice. In comparison, G3:Fab1-HK129-LL157-BiFolate-Bi5KPEG (dual PEG composition) had a TGI of 76%, with 3 of 10 animals experiencing CR (Figure 20A). All test substances were well tolerated, with G3 (dual PEG composition) showing slight weight loss in a few mice, but these did not exceed a 15% loss (Figure 20B).

[0431] Biomarker Analysis: Blood samples were collected from each animal on days 7 and 14 after the initiation of treatment, as outlined above. Blood samples were analyzed for...

Claims

1. a) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 32; or b) a light chain comprising the amino acid sequence of SEQ ID NO: 60, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 3; or c) a light chain comprising the amino acid sequence of SEQ ID NO: 60, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 31; 1. An anti-CD3 antibody or antigen-binding fragment comprising: an anti-CD3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is at position 129 of SEQ ID NO: 32, 3, or 31, numbered according to the Kabat numbering system, wherein the non-naturally encoded amino acid is para-acetylphenylalanine, and wherein the non-naturally encoded amino acid is linked to folic acid.

2. a) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 3; or b) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; or c) a light chain comprising the amino acid sequence of SEQ ID NO: 61 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34; or d) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; 1. An anti-CD3 antibody or antigen-binding fragment comprising: an anti-CD3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a non-naturally encoded amino acid, wherein the non-naturally encoded amino acid is at position 129 of SEQ ID NO: 3, 33, 34, or 35, numbered according to the Kabat numbering system, wherein the non-naturally encoded amino acid is para-acetylphenylalanine, and wherein the non-naturally encoded amino acid is linked to folic acid.

3. a) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 32; or b) a light chain comprising the amino acid sequence of SEQ ID NO: 60, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 3; or c) a light chain comprising the amino acid sequence of SEQ ID NO: 60, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 31; 1. An anti-CD3 antibody or antigen-binding fragment comprising: an anti-CD3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises two non-naturally encoded amino acids; one non-naturally encoded amino acid is at position 129 of SEQ ID NO: 32, 3, or 31 and one non-naturally encoded amino acid is at position 157 of SEQ ID NO: 59 or 60, numbered according to the Kabat numbering system, wherein each non-naturally encoded amino acid is para-acetylphenylalanine, and each non-naturally encoded amino acid is linked to folic acid.

4. a) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 3; or b) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 33; or c) a light chain comprising the amino acid sequence of SEQ ID NO: 61 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 34; or d) a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; 1. An anti-CD3 antibody or antigen-binding fragment comprising: an anti-CD3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises two non-naturally encoded amino acids; one non-naturally encoded amino acid is at position 129 of SEQ ID NO: 3, 33, 34, or 35 and one non-naturally encoded amino acid is at position 157 of SEQ ID NO: 59 or 61, numbered according to the Kabat numbering system, wherein each non-naturally encoded amino acid is para-acetylphenylalanine, and each non-naturally encoded amino acid is linked to folic acid.

5. a light chain comprising the amino acid sequence of SEQ ID NO: 59, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 32; The anti-CD3 antibody or antigen-binding fragment of claim 1 or 3.

6. The antibody or antigen-binding fragment may be an IgG, Fab, or (Fab') ２ The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 5, comprising:

7. 7. The anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 6, wherein the non-naturally encoded amino acid, or each non-naturally encoded amino acid, is linked to a linker, a polymer, or a combination thereof.

8. The anti-CD3 antibody or antigen-binding fragment of claim 7 , wherein the linker is a bifunctional linker.

9. The anti-CD3 antibody or antigen-binding fragment of claim 7 , wherein the polymer is a water-soluble polymer.

10. 10. The anti-CD3 antibody or antigen-binding fragment of any one of claims 1-9, wherein the non-naturally encoded amino acid, or each non-naturally encoded amino acid, is linked to both folate and a water soluble polymer using a bifunctional linker.

11. The non-naturally encoded amino acid or each non-naturally encoded amino acid is linked to a bifunctional water soluble polymer-folate linker having the structure: 【Chemical 1】 where A has the structure: 【Chemistry 2】 B is a divalent group linking A and C, having the structure: 【Chemistry 3】 C and E are each independently -alkylene-; -alkylene-C(O)-, -(alkylene-O) ｎ’ -Alkylene-, -(alkylene-O) ｎ’ -alkylene-C(O)-, and -(alkylene-O) ｎ - (CH ２ ) ｎ’ -NHC(O)-(CH ２ ) ｎ’ -C(Me) ２ -S-S-(CH ２ ) ｎ’ —NHC(O)—(alkylene-O) ｎ -alkylene-, wherein n' is independently an integer of 1 or greater; D is a trivalent group connecting C, F, and E; F is the water-soluble polymer; and Y is hydroxylamine (-ONH ２ ), methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, ketoamine, ketoalkyne, alkyne, cycloalkyne, and enedione.

12. 12. The anti-CD3 antibody or antigen-binding fragment of claim 11, wherein the non-naturally encoded amino acid or each non-naturally encoded amino acid is linked to the bifunctional water-soluble polymer-folate linker via an oxime bond.

13. The water-soluble polymer comprises one or more poly(ethylene glycol) (PEG) molecules. The anti-CD3 antibody or antigen-binding fragment of any one of claims 9 to 12.

14. 14. The anti-CD3 antibody or antigen-binding fragment of claim 13, wherein the one or more PEG molecules are linear.

15. The anti-CD3 antibody or antigen-binding fragment of claim 13 or 14, wherein the average molecular weight of the one or more PEG molecules is 5 kDa to 20 kDa.

16. 15. The anti-CD3 antibody or antigen-binding fragment of claim 13 or 14, wherein the average molecular weight of the one or more PEG molecules is 10 kDa.

17. each non-naturally encoded amino acid is linked to both a folate and a water soluble polymer using a bifunctional linker; the water-soluble polymer comprises a PEG molecule; The PEG molecule is linear, and The average molecular weight of the PEG molecules is 10 kDa. The anti-CD3 antibody or antigen-binding fragment of claim 3.

18. each non-naturally encoded amino acid is linked to both a folate and a water soluble polymer using a bifunctional linker; the water-soluble polymer comprises a PEG molecule; The PEG molecule is linear, and The average molecular weight of the PEG molecules is 10 kDa. The anti-CD3 antibody or antigen-binding fragment of claim 4.

19. A pharmaceutical composition for therapeutic use, comprising the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 18.

20. A pharmaceutical composition for treating a disease or condition in cells that highly express folate receptors, comprising an anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 18.

21. 21. The pharmaceutical composition of claim 20, wherein the number of folate receptors is 10,000 or more.

22. A pharmaceutical composition for treating cancer, comprising the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 18.

23. 23. The pharmaceutical composition of claim 22, wherein the cancer is characterized by high expression of folate receptor alpha (FOLR1).

24. 24. The pharmaceutical composition of claim 23, wherein the cancer is ovarian cancer.

25. 25. The pharmaceutical composition of claim 24, wherein the ovarian cancer comprises an epithelial tumor, a stromal tumor, a germ cell tumor, a fallopian tube cancer, or a primary peritoneal carcinoma.

26. A pharmaceutical composition comprising a therapeutically effective amount of the anti-CD3 antibody or antigen-binding fragment of any one of claims 1 to 18 and a pharmaceutically acceptable carrier or excipient.

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