TWI827807B - Anti-cd3 antibody folate bioconjugates and their uses - Google Patents

Abstract

Described herein are novel anti-CD3 Folate antibodies and uses thereof in the treatment of diseases or conditions that would benefit from such.

Description

Anti-CD3 antibody folate bioconjugates and their uses

The present disclosure relates to the field of immuno-oncology. More specifically, the present invention refers in particular to an anti-CD3 antibody conjugated to one or more folate molecules and fragments or variants thereof. The invention also relates to anti-CD3 Fab folate antibodies conjugated to polyethylene glycol (PEG) and variants thereof.

Ovarian cancer is one of the most common cancers in women worldwide. Approximately 250,000 women will be diagnosed with ovarian cancer each year, and approximately 140,000 women will die from the disease each year. The five-year survival rate for ovarian cancer varies based on the type and stage of the cancer, with the trend being that the more advanced the ovarian cancer is, the worse the five-year survival rate is. Current treatment options for ovarian cancer include chemotherapy, surgery, radiation, or a combination of these therapies. After surgical resection followed by platinum-based chemotherapy, the response rate for advanced ovarian cancer is 80%, with a complete response rate of 40-60%. Unfortunately, approximately 70% of these patients will relapse, with a median progression-free survival of 18 months.

Currently, there is a shortfall in the area of therapies targeting ovarian cancer and ovarian cancer types, including epithelial, stromal, and germ cell tumors and including fallopian tube and primary peritoneal cancers. Although surgery, radiation, and various chemotherapy treatments are commonly used to treat ovarian cancer patients, the U.S. Food and Drug The Food and Drug Administration (FDA) recently accepted a supplemental biologics license application (sBLA) for bevacizumab (Avastin) as first-line therapy for the treatment of advanced ovarian cancer.

Immunotherapy is being evaluated as a new treatment option for cancer patients, which uses biological or engineered T cells to stimulate a patient's immune system to attack their cancer. Several immunotherapies have shown great promise and have been approved to treat various types of cancer. Immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T), bispecific T cell adapter proteins (BiTE), various designed T cell-dependent bispecific antibodies (TDB), NK cell-dependent bispecific antibodies Specific antibodies, macrophage-dependent bispecific antibodies, and autologous antigen-presenting cells (APC)/cancer vaccines are some of the immunotherapies used to treat cancer patients.

Some biologics undergoing clinical evaluation in patients with ovarian cancer include an antibody-drug conjugate directed against folate receptor alpha (Mirvetuximab soravtansine (IMGN853)), an EpCAM-CD3 bispecific antibody (Catumaxomab), DLL4-VEGF bispecific, vaccine targeting NY-ESO-1 and CAR-T targeting folate receptor alpha (FOLR1).

Ovarian cancer has been reported to have an immunosuppressive environment, making the development of certain types of immunotherapies such as checkpoint inhibitors a challenge. There are currently no immunotherapies approved to treat patients with ovarian cancer, but some are being evaluated in clinical trials.

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 human folate receptor (FR) family includes FRα, FRβ and FRγ. Folate receptor alpha (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 folate's main metabolite, 5-methyl-tetrahydrofolate (5-MTHF). High expression of FOLR1 was observed in approximately 76% of high-grade serous ovarian cancers (the predominant histotype), compared with 11% of mucinous ovarian cancers. FOLR1 Expression is low and restricted in normal tissues, making it a good target for oncology drug development efforts.

To overcome deficiencies in the art, the inventors have developed an antibody conjugate that incorporates one or more non-naturally encoded amino acids in an anti-CD3 antibody and also contains one or more folate molecules. Bispecific antibodies can comprise or consist of anti-CD3 antibodies that have been engineered to have one or more Fab (fragment antigen binding) heavy or light chains. Non-naturally encoded amino acids. Bispecific antibodies or antibody fragments may comprise anti-CD3 antibodies that have been engineered to have one or more non-naturally encoded amino acids on the heavy and/or light chain.

Anti-CD3 bispecific antibodies that recruit cytotoxic T cells to cancer cells are a promising new approach for treating a variety of liquid and solid tumors. In one aspect, the invention provides an anti-CD3 antibody. In one embodiment, the anti-CD3 antibody is a bispecific antibody. In one embodiment, the anti-CD3 antibody comprises anti-CD3 Fab. In one embodiment, the anti-CD3 antibody contains a non-naturally encoded amino acid in the heavy or light chain of the Fab, preferably in the heavy chain. In one embodiment, the non-naturally encoded amino acid is para-acetyl phenylalanine, where "phenylalanine" is abbreviated as F, so "para-acetyl phenylalanine" is abbreviated as Paf; as mentioned in the present invention The English abbreviation of "phenylalanine" is represented by F, which will be explained first). In one embodiment, the anti-CD3 antibody contains two or more non-naturally encoded amino acids in the heavy chain and/or light chain of the Fab, such as two, three or four non-naturally encoded amino acids. acid, optionally wherein two or more of the non-naturally encoded amino acids are pAF. In one embodiment, the anti-CD3 Fab is conjugated with folic acid. In one embodiment, the anti-CD3 Fab is conjugated with a water-soluble polymer such as polyethylene glycol (PEG). In one embodiment, a bifunctional graft is optionally used The anti-CD3 Fab was first conjugated with both folic acid and polyethylene glycol (PEG). In one embodiment, the anti-CD3 Fab is conjugated to two folate and two polyethylene glycol (PEG) molecules. In one embodiment, conjugation is via the side chain of a non-naturally encoded amino acid, such as pAF. In one embodiment, anti-CD3 antibodies recruit cytotoxic T cells to folate receptor positive (FR+) tumor cells. In one embodiment, the anti-CD3 antibody has improved efficacy, reduced toxicity, improved pharmacokinetic (PK) properties, improved affinity, improved tumor-associated antigen (TAA) binding, improved in vivo half-life (T1/ 2), improved in vitro activity, improved serum half-life and/or improved in vivo activity. In one embodiment, anti-CD3 antibodies have improved efficacy. In one embodiment, the anti-CD3 antibody has reduced toxicity. In one embodiment, the anti-CD3 antibody has improved PK properties. In one embodiment, the anti-CD3 antibody has improved affinity. In one embodiment, the anti-CD3 antibody has improved TAA binding. In one embodiment, the anti-CD3 antibody has improved T1/2 in vivo. In one embodiment, the anti-CD3 antibody has improved in vitro activity. In one embodiment, the anti-CD3 antibody has improved serum half-life. In one embodiment, the anti-CD3 antibody has improved in vivo activity. The present invention provides optimization of anti-CD3 Fab-folate conjugates that target cytotoxic T cells to folate receptor-positive (FR+) tumor cells for optimal efficacy, reduced toxicity, and optimal drug metabolism. Kinetic (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 for optimal efficacy, reduced toxicity, and optimal Pharmacokinetic (PK) properties. To achieve an optimal balance between efficacy and toxicity due to cytokine release syndrome (CRS), the inventors fine-tuned the affinity of the anti-CD3 antibody and optimized tumor-associated antigen (TAA) binding. To increase the in vivo half-life (T1/2), folic acid and PEG molecules of various sizes were simultaneously conjugated by using a bifunctional linker. Optimized conjugates showed potent and selective in vitro activity, good serum half-life, and potent in vivo activity in xenograft mouse models. This semisynthetic approach may be suitable for generating additional anti-CD3 bispecific reagents using small molecule ligands selective for other TAAs.

From another aspect, the present invention provides anti-CD3 antibodies and their conjugates with folic acid. The invention also provides anti-CD3 antibodies and their conjugates with PEG. The invention also provides anti-CD3 antibodies and their conjugates with folic acid and PEG. In one embodiment, the novel anti-CD3 antibodies of the invention comprise one or more non-naturally encoded amino acids. In one embodiment, the anti-CD3 antibody comprises an intact antibody heavy chain. In one embodiment, the anti-CD3 antibody comprises an intact antibody light chain. In one embodiment, the anti-CD3 antibody comprises an intact antibody heavy chain and an intact antibody light chain. In one embodiment, the anti-CD3 antibody comprises the variable region of an antibody light chain. In one embodiment, the anti-CD3 antibody comprises the variable region of an antibody heavy chain. In one embodiment, the anti-CD3 antibody comprises a variable region of a light chain and a variable region of a heavy chain. In one embodiment, the anti-CD3 antibody comprises at least one CDR of the antibody light chain. In one embodiment, the anti-CD3 antibody comprises at least one CDR of the anti-CD3 antibody heavy chain. In one embodiment, the anti-CD3 antibody comprises at least one CDR of the light chain and at least one CDR of the heavy chain. In one embodiment, the anti-CD3 antibody contains three CDRs of the light chain. In one embodiment, the anti-CD3 antibody contains three CDRs of the heavy chain. In one embodiment, the anti-CD3 antibody comprises three CDRs of the light chain and three CDRs of the heavy chain. In one embodiment, the anti-CD3 antibody comprises a Fab. In one embodiment, the anti-CD3 antibody contains two Fabs. In one embodiment, the anti-CD3 antibody contains two or more Fabs. In one embodiment, the anti-CD3 antibody includes scFv (single-chain variable fragment, single-chain variable fragment). In one embodiment, the anti-CD3 antibody contains two scFv. In one embodiment, the anti-CD3 antibody contains two or more scFvs. In one embodiment, the anti-CD3 antibodies comprise minibodies. In one embodiment, the anti-CD3 antibody includes two minibodies. In one embodiment, the anti-CD3 antibody comprises two or more minibodies. In one embodiment, the anti-CD3 antibody comprises a diabody antibody. In one embodiment, the anti-CD3 antibody includes two diabody antibodies. In one embodiment, the anti-CD3 antibody comprises two or more diabody antibodies. In one embodiment, the anti-CD3 antibody includes BiTE (Bi-specific T-cell engagers, bispecific T-cell engagers). In one embodiment, the anti-CD3 antibody contains two BiTEs. In one embodiment, the anti-CD3 antibody contains two or more BiTEs. In one embodiment, the anti-CD3 antibody comprises DART. In one embodiment, the anti-CD3 antibody includes two DARTs (Dual-affinity Re-targeting Antibody). In one embodiment, anti-CD3 antibodies Contains two or more DARTs. In one embodiment, the anti-CD3 antibody includes TandAb (Tandem Diabody). In one embodiment, the anti-CD3 antibody comprises two TandAbs. In one embodiment, the anti-CD3 antibody comprises two or more TandAbs. In one embodiment, the anti-CD3 antibody comprises a variable region of a light chain and a variable region of a heavy chain. In one embodiment, the anti-CD3 antibody comprises an intact light chain and an intact heavy chain. In one embodiment, the anti-CD3 antibody includes one or more Fc (Fragment crystallizable region) domains or portions thereof. In one embodiment, the anti-CD3 antibody includes a combination of any of the above embodiments. In one embodiment, the anti-CD3 antibody comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments. In one embodiment, an anti-CD3 antibody comprises a polypeptide that binds a binding partner, wherein the binding partner includes an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance. In one embodiment, the anti-CD3 antibody is associated with a non-antibody scaffold molecule or substance.

In one embodiment, the anti-CD3 antibody contains one or more post-translational modifications. In one embodiment, the anti-CD3 antibody is linked to a linker, polymer, or bioactive molecule. In one embodiment, the anti-CD3 antibody is linked to a bifunctional polymer, a bifunctional linker, or at least one additional anti-CD3 antibody. In one embodiment, the anti-CD3 antibody is linked to a polypeptide that is not an anti-CD3 antibody. In one embodiment, an antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides that may also comprise a non-naturally encoded amino acid. In one embodiment, an antigen-binding polypeptide comprising a non-naturally encoded amino acid is linked to one or more polypeptide small molecule conjugates that may also comprise a non-naturally encoded amino acid. In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides that may also comprise a non-naturally encoded amino acid.

In one embodiment, the non-naturally encoded amino acid is linked to a small molecule ligand. In one embodiment, the non-naturally encoded amino acid is linked to two small molecule ligands. In one embodiment, the non-naturally encoded amino acid is linked to two or more small molecule ligands. In one embodiment, the small molecule ligand includes folic acid or DUPA (2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, 2-[3-(1,3-dicarboxypropyl)ureido] glutaric acid) molecule. In one embodiment, the small molecule ligand includes two folates or two DUPA molecules. son. In one embodiment, the small molecule ligand includes two or more folic acid or two or more DUPA molecules. In one embodiment, the non-naturally encoded amino acid is linked to a water-soluble polymer. In one embodiment, the water-soluble polymer contains polyethylene glycol moieties. In one embodiment, the polyethylene glycol molecule is a bifunctional polymer. In one embodiment, the bifunctional polymer is linked to a second polypeptide. In one embodiment, the second polypeptide is an antigen-binding polypeptide. In one embodiment, the second polypeptide is an anti-CD3 antibody. In one embodiment, the small molecule ligand is attached to a water-soluble polymer. In one embodiment, two small molecule ligands are attached to two water-soluble polymers. In one embodiment, two or more small molecule ligands are attached to two or more water-soluble polymers. In one embodiment, folic acid is linked to a PEG molecule. In one embodiment, two folate molecules are linked to two water-soluble polymers. In one embodiment, two or more folate molecules are linked to two or more PEG molecules.

In one embodiment, the amino acid substitutions in the anti-CD3 antibody may be by naturally occurring or non-naturally occurring amino acids, provided that at least one substitution is by a non-naturally encoded amino acid.

In one embodiment, the non-naturally encoded amino acid includes a carbonyl group, an acetyl group, an amineoxy group, a hydrazine group, a hydrazine group, a semicarbazide group, an azide group, or an alkynyl group.

In one embodiment, the polyethylene glycol molecules have an average molecular weight of about 0.1 kDa to about 100 kDa. In one embodiment, the average molecular weight of polyethylene glycol molecules ranges from 0.1 kDa to 50 kDa. In one embodiment, the average molecular weight of polyethylene glycol is 1 kDa to 25 kDa, or 2 kDa to 22 kDa, or 5 kDa to 20 kDa. For example, the average molecular weight may be about 5 kDa or about 10 kDa or about 20 kDa. For example, the polyethylene glycol polymer may have an average molecular weight of 5 kDa or 10 kDa or 20 kDa. In certain embodiments, molecular weight is determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, mass spectrometry, and capillary electrophoresis.

In one embodiment, the polyethylene glycol molecule is a branched polymer. In one embodiment, the molecular weight of each branch of the polyethylene glycol branched polymer is 1 kDa to 100 kDa, or 1 kDa to 50 kDa. In one embodiment, the molecular weight of each branch of the polyethylene glycol branched polymer is 1 kDa to 25 kDa, or 2kDa to 22kDa, or 5kDa to 20kDa. For example, the molecular weight of each branch of the polyethylene glycol branched polymer may be about 5 kDa or about 10 kDa or about 20 kDa. For example, the molecular weight of each branch of the polyethylene glycol branched polymer may be 5 kDa or 10 kDa or 20 kDa.

From a further aspect, the invention also provides an anti-CD3 antibody polypeptide comprising a linker, polymer or bioactive molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acid Amino acids are incorporated into the polypeptide by ribosomes at preselected positions. Embodiments of the invention provide anti-CD3 antibodies comprising at least one of SEQ ID NOs: 1-62. The anti-CD3 antibody includes two of SEQ ID NOs: 1-62. Embodiments of the present invention provide anti-CD3 antibodies comprising two of SEQ ID NOs: 1-62. The anti-CD3 antibody comprises any one of SEQ ID NO: 1-6 and any one of SEQ ID NO: 7-9. Embodiments of the present invention provide anti-CD3 Fab antibodies comprising both of SEQ ID NOs: 1-5. In other embodiments, the invention provides a bispecific binding molecule comprising (1) a first binding domain and (2) a second binding domain, wherein the second binding domain is selected from SEQ ID NO. :1-62. In other embodiments, the invention provides a bispecific binding molecule comprising (1) a first binding domain and (2) a second binding domain, wherein the second binding domain comprises anti-CD3, The anti-CD3 includes SEQ ID NO: 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, Any of 53, 54, 55, 56 and 57 and any of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61 and 62. In other embodiments, the invention provides a bispecific binding molecule comprising (1) a first binding domain and (ii) a second binding domain, wherein the second binding domain comprises an anti-CD3 Fab, The anti-CD3 Fab includes SEQ ID NO: 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, Any of 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56 and 57. In other embodiments, the invention provides a bispecific binding molecule comprising (1) a first binding domain and (2) a second binding domain, wherein the second binding domain comprises an anti-CD3 Fab, The anti-CD3 Fab includes SEQ ID NO: 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, Any of 52, 53, 54, 55, 56 and 57; and any of SEQ ID NOs: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61 and 62 . In other embodiments, the present invention provides a cytotoxic active CD3 specific binding construct comprising the amino acid sequence shown in one or more of SEQ ID NOs: 1-62. In other embodiments, the present invention provides a cytotoxic active CD3 specific binding construct comprising the amino acid sequences shown in both of SEQ ID NOs: 1-62. In other embodiments, the invention provides a cytotoxic active CD3 specific binding construct comprising anti-CD3 comprising SEQ ID NO: 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 , any of 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56 and 57. In other embodiments, the invention provides a cytotoxic active CD3 Fab specific binding construct comprising an anti-CD3 Fab comprising SEQ ID NOs: 7, 8, 9, 18, 19, 20, Any of 39, 58, 59, 60, 61 and 62. In other embodiments, the invention provides a cytotoxic active CD3 specific binding construct comprising anti-CD3 comprising SEQ ID NO: 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, Any of 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56 and 57; and SEQ ID NO: 7, 8, 9, 18, 19, 20, 39, Any of 58, 59, 60, 61 and 62. Another embodiment of the invention provides an anti-CD3 Fab antibody, wherein the anti-CD3 antibody comprises a binding domain comprising (a) a VH (heavy chain variable region) domain selected from SEQ. The amino acid sequences of ID NOs: 1-6, and (b) a VL (variable light chain) domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-9.

In yet another aspect, another embodiment of the invention provides an anti-CD3 antibody, wherein the antibody comprises one or more post-translational modifications. Embodiments of the invention provide an anti-CD3 Fab antibody, wherein the antibody is linked to a linker, polymer or bioactive molecule. Another embodiment of the invention provides an anti-CD3 antibody, wherein the biologically active molecule is folic acid. Embodiments of the invention provide anti-CD3 antibodies comprising one or more folates. Embodiments of the invention provide anti-CD3 antibodies comprising two folates. Another embodiment of the invention provides anti-CD3 antibodies, wherein the biologically active molecule is DUPA. Embodiments of the invention provide anti-CD3 antibodies comprising one or more DUPA. Embodiments of the invention provide anti-CD3 antibodies comprising two DUPAs.

In certain embodiments, the anti-CD3 antibody comprises SEQ. ,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 , the heavy chain amino acid sequence of any one of 50, 50, 51, 52, 53, 54, 55, 56 and 57; and SEQ.ID.NO: 7, 8, 9, 18, 19, 20, The light chain amino acid sequence of any of 39, 58, 59, 60, 61 and 62.

In one embodiment, the anti-CD3 antibody includes: SEQ ID NO: 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, Anti-CD3 for the amino acid sequence of any of 54, 55, 56 and 57. In one embodiment, the anti-CD3 antibody comprises: an amino acid sequence comprising any one of SEQ ID NO: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61 and 62 of anti-CD3. In one embodiment, the anti-CD3 antibody includes: SEQ ID NO: 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 , an anti-CD3 Fab containing the amino acid sequence of any one of 50, 50, 51, 52, 53, 54, 55, 56 and 57 and comprising SEQ ID NO: 7, 8, 9, 18, 19, 20, Anti-CD3 Fab of the amino acid sequence of any one of 39, 58, 59, 60, 61 and 62. Another embodiment of the invention provides anti-CD3 variants comprising the heavy chain of SEQ ID NO: 1-5 and the light chain of SEQ ID NO: 7-9. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising 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 one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising 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 contains at least one non-natural Amino acids. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising 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 contains two non-natural Amino acids. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising SEQ ID NO: 7, which comprises at least one unnatural amino acid; and SEQ ID NO: 10, or 14, or 11, or One of 15, or 12, or 16, or 13, or 17, or 1, or 18, or 19, which contains at least one non-natural amino acid. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising SEQ ID NO: 12, which comprises at least one unnatural amino acid; and one of SEQ ID NO: 18, or 19, or 16, which contains at least one non-natural amino acid. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising SEQ ID NO: 58 and 49, or 58 and 40, or 59 and 50, or 59 and 41, or 59 and 42, or 59 and 52, or 59 and 43, or 59 and 44, or 59 and 45, or 59 and 54, or 59 and 55, or 59 and 46, or 9 and 44, or 9 and 53, or 60 and 44, or 60 and 53, or 60 and 42, or 60 and 51, 60 and 50, or 60 and 41, or 61 and 45, or 61 and 54, or 62 and 56, or 62 and 47, or 62 and 48, or 62 and 57, or 7 and 47, or 7 and 56. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing two unnatural amino acids, the antibody comprising SEQ ID NOs: 18 and 16, or 18 and 59, or 18 and 43.

In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing SEQ ID NO: 1 and 7, or 1 and 8, or 1 and 9. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing SEQ ID NO: 2 and 7, or 2 and 8, or 2 and 9. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing SEQ ID NO: 3 and 7, or 3 and 8, or 3 and 9. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing SEQ ID NO: 4 and 7, or 4 and 8, or 4 and 9. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing SEQ ID NO: 5 and 7, or 5 and 8, or 5 and 9. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab containing SEQ ID NO: 6 and 7, or 6 and 8, or 6 and 9. The anti-CD3 antibody can be a bispecific antibody comprising (1) a first binding domain and (2) a second binding domain, wherein the second binding domain comprises the anti-CD3 Fab. Other embodiments of the invention provide anti-CD3 Fab antibodies selected from SEQ. ID. NO: 1 to 62, into which non-naturally encoded amino acids are incorporated. In one embodiment, non-naturally encoded amino acids are site-specifically incorporated into the antibody.

In other embodiments, the invention provides anti-CD3 Fab antibodies, wherein the non-naturally encoded amino acid is selected from: O -methyl-L-tyrosine, L-3- (2-naphthyl)alanine (L-3-(2-naphthyl)alanine), 3-methyl-phenylalanine (3-methyl-phenylalanine), O-4-allyl-L-tyrosine ( o -4-allyl-L-tyrosine), 4-propyl-L-tyrosine, p-propargyloxy-L-phenylalanine, tri-O-acetyl GlcNAc-serine, L-DOPA, fluorinatedphenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine p -azido-L-phenylalanine), p-acylphenylalanine (p-acylphenylalanine), p-benzoyl-L-phenylalanine ( p -benzoyl-L-phenylalanine), L-phosphoserine ( L-phosphoserine), phosphonoserine, phosphonotyrosine, p -iodo-phenylalanine, p -bromophenylalanine, p-amino- L- amino -L-phenylalanine or isopropyl-L-phenylalanine.

In yet another aspect, another embodiment of the invention provides anti-CD3 Fab antibodies wherein non-naturally encoded amino acids are position-specifically incorporated. Embodiments of the invention provide an anti-CD3 Fab antibody in which non-naturally encoded amino acids are position-specifically incorporated at positions H114, H115, H129, L157, H160, L172 and L205 (according to those skilled in the art) anywhere in the well-known Kabat number). Embodiments of the invention provide anti-CD3 Fab antibodies in which non-naturally encoded amino acids are position-specifically incorporated at positions H114, H115, H129, L157, H160, L172, and L205 (according to Kabat numbering). Embodiments of the present invention provide an anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 114. Embodiments of the invention provide an anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 115. Embodiments of the invention provide an anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 129. Embodiments of the invention provide an anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 157. Embodiments of the present invention provide an anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 160. Embodiments of the invention provide an anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 172. Embodiments of the present invention provide a An anti-CD3 Fab antibody in which a non-naturally encoded amino acid is position-specifically incorporated at position 205. Another embodiment of the invention provides anti-CD3 Fab antibodies into which at least one non-naturally encoded amino acid is incorporated. Another embodiment of the invention provides anti-CD3 Fab antibodies in which two non-naturally encoded amino acids are incorporated. Other embodiments of the invention provide an anti-CD3 Fab variant comprising at least one non-naturally encoded amino acid in the heavy or light chain. Other embodiments of the invention provide an anti-CD3 Fab antibody comprising a non-naturally encoded amino acid in the light chain. Other embodiments of the invention provide an anti-CD3 Fab antibody comprising a non-naturally encoded amino acid in the heavy chain. Another embodiment of the invention provides an anti-CD3 Fab variant comprising two non-naturally encoded amino acids.

From yet another perspective, 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. Embodiments of the present invention provide an anti-CD3 Fab variant wherein the amino acid extension includes the amino acid DKTHT. Another embodiment of the invention provides an anti-CD3 Fab variant further comprising an amino acid extension at the C-terminus of the heavy chain. Other embodiments of the invention provide an anti-CD3 Fab variant wherein the amino acid extension includes the amino acid DKTHT.

Another embodiment of the invention provides anti-CD3 Fab antibodies, wherein the heavy chain sequence contains mouse framework residues at one or more positions for antigen binding. Other embodiments of the invention provide anti-CD3 Fab antibodies, wherein the heavy chain sequence comprises mouse framework residues at position 30, 49, 77 or 93 (according to Kabat numbering). Another embodiment of the invention provides anti-CD3 Fab antibodies, wherein the light chain sequence contains mouse framework residues at one or more positions. Another embodiment of the invention provides an anti-CD3 Fab antibody, wherein the light chain sequence comprises framework residues at position 36, 46, 49, 57 or 58 (according to Kabat numbering).

In some embodiments of the invention, anti-CD3 Fab antibodies include linkers. In another embodiment, the invention provides an anti-CD3 Fab antibody, wherein the linker is a water-soluble polymer. Another embodiment of the invention provides an anti-CD3 Fab antibody, wherein the water-soluble polymer comprises polyethylene glycol. Other embodiments of the invention provide an anti-CD3 Fab antibody, wherein the water-soluble polymer is linear or branched. Another embodiment of the invention provides anti-CD3 Fab antibodies, wherein a water-soluble polymer is linked to a non-naturally encoded amino acid present in the antibody. In one embodiment, the linker is attached to the anti-CD3 Fab antibody via the side chain of a non-natural amino acid. Embodiments of the invention provide an anti-CD3 Fab antibody comprising at least two amino acids linked to a water-soluble polymer comprising polyethylene glycol. Another embodiment of the invention provides an anti-CD3 Fab antibody, wherein at least one amino acid linked to the water-soluble polymer is a non-naturally encoded amino acid. Another embodiment of the invention provides anti-CD3 Fab antibodies comprising one or more polyethylene glycols. Another embodiment of the invention provides anti-CD3 Fab antibodies, wherein the polyethylene glycol is 1 kDa to 100 kDa. Another embodiment of the invention provides an anti-CD3 Fab antibody comprising one or more folic acid and one or more polyethylene glycols. Another embodiment of the invention provides an anti-CD3 Fab antibody comprising two folic acids and two polyethylene glycols.

From yet another perspective, in another embodiment, the present invention provides a method of optimizing cell killing in cells expressing a high number of folate receptors, said method comprising an anti-CD3 Fab antibody, wherein said antibody comprises a or A plurality of folates; and wherein one or more non-naturally encoded amino acids are incorporated into the antibody. In another embodiment, the invention provides a method for optimizing the killing of tumor cells expressing a high number of folate receptors by redirected T cells, the method comprising an anti-CD3 Fab antibody, wherein the antibody comprises one or more folic acid; and wherein one or more non-naturally encoded amino acids are incorporated into the antibody. In another embodiment, the method further includes one or more water-soluble polymers. Another embodiment of the invention provides a method wherein the water-soluble polymer comprises polyethylene glycol. In another embodiment a method is provided wherein the water-soluble polymer is linear or branched. polyethylene glycol 1kDa to 100kDa. Polyethylene glycol is 5, 10, 20, 30, 40, 50 or 60 kDa. In another embodiment, the number of folate receptors is equal to or greater than 10,000.

From yet another perspective, another embodiment of the invention provides a method of reducing cytotoxicity in a cell. Another embodiment of the invention provides a method of reducing cytotoxicity in cells by optimizing or reducing the affinity of an anti-CD3 antibody for its target for T cell recruitment. Another embodiment of the invention provides a method of increasing the cytotoxicity of tumor cells by conjugating more than one folate prior to T cell binding to preferentially bind to tumor cells. Another embodiment of the invention provides a method of improving the serum half-life of anti-CD3 Fab antibodies. Another embodiment of the invention provides a method of improving the serum half-life of an anti-CD3 Fab antibody by conjugating the Fab with a pharmacokinetic (PK) extending molecule, including but not limited to HSA, C12-C16 acyl chains, XTEN or water-soluble polymers such as PEG. Another embodiment provides a method of recruiting cytotoxic T cells to FR+ tumor cells. Another embodiment provides a method for improving efficacy, reducing toxicity, improving PK properties, improving affinity, improving TAA binding, improving T1/2 in vivo, improving activity in vitro, improving serum half-life, and/or improving activity in vivo. Another embodiment provides a method of improving efficacy. Another embodiment provides a method of reducing toxicity. Another embodiment provides a method of improving PK properties. Another embodiment provides a method for improving affinity. Another embodiment provides a method of improving TAA binding. Another embodiment provides a method of improving T1/2 in vivo. Another embodiment provides a method of improving in vitro activity. Another embodiment provides a method for improving activity in vivo.

In yet another aspect, another embodiment of the invention provides an anti-CD3 Fab antibody, wherein the heavy or light chain sequence contains a human germline mutation at one or more positions. Embodiments of the invention provide an anti-CD3 Fab antibody, wherein the human germline mutation of the heavy chain sequence is at position 35 or 52 (according to Kabat numbering). Another embodiment of the invention provides an anti-CD3 Fab antibody, wherein the human germline mutation of the light chain sequence is at position 53 (according to Kabat numbering).

Another embodiment of the invention provides an anti-CD3 Fab antibody comprising a PEG folate linker having a structure according to compounds 29A, 29B, 29C, 29D, 29E, 30A, 30B, 30C, 30D and 30E.

From yet another perspective, in another embodiment, the invention provides a method of treating a patient having a disease or disorder in cells expressing a high number of folate receptors, said method comprising administering to said patient a therapeutically effective amount The anti-CD3 Fab antibodies disclosed in the present invention. In another embodiment of the invention, a bispecific anti-CD3 Fab comprising two folate and two pegylated molecules is provided. In another embodiment, the bispecific anti-CD3 Fab antibody further comprises a non-naturally encoded amino acid incorporated site specifically. The invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. In one embodiment, the cancer is ovarian cancer. In one embodiment, the ovarian cancer is an epithelial, stromal and germ cell tumor. In one embodiment, the ovarian cancer includes fallopian tube cancer and primary peritoneal cancer. In one embodiment, the cancer is characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. In one embodiment, cancer is treated by recruiting cytotoxic T cells to folate receptor positive (FR+) tumor cells. The present invention provides a method for treating genetic diseases by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The present invention provides a method of treating AIDs by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The present invention provides a method of treating diabetes by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the present invention. The anti-CD3 antibody of the invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is conjugated to two folate and two pegylated molecules. In another example, a non-naturally encoded amino acid is site-specifically incorporated into the anti-CD3 Fab antibody and linked via the side chain of the non-natural amino acid to two folates and two polyethylene glycol. Alcoholized molecular conjugation.

The anti-CD3 antibodies of the invention are useful in treating diseases or conditions in cells expressing high numbers of folate receptors. The anti-CD3 antibodies of the invention are used to treat cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the ovarian cancer is an epithelial, stromal, and germ cell tumor. In one embodiment, ovarian cancer includes fallopian tube cancer and primary peritoneal cancer. In one embodiment, the cancer is characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. In one embodiment, 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 antibody of the present invention is used to treat AIDS. The anti-CD3 antibodies of the present invention are used to treat diabetes. The anti-CD3 antibody of the invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is conjugated to two folate and two pegylated molecules. In another example, a non-naturally encoded amino acid is site-specifically incorporated into the anti-CD3 Fab antibody and conjugation to two folate and two PEGylated molecules is via the non-natural amine group The side chain of the acid is realized. The anti-CD3 antibodies of the invention can be used in the manufacture of medicaments for the treatment of diseases or conditions in cells expressing high numbers of folate receptors. The anti-CD3 antibodies of the present invention can be used to manufacture drugs for treating cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, the ovarian cancer is an epithelial, stromal, and germ cell tumor. In one embodiment, ovarian cancer includes fallopian tube cancer and primary peritoneal cancer. In one embodiment, the cancer is characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. In one embodiment, cancer is treated by recruiting cytotoxic T cells to folate receptor positive (FR+) tumor cells. The anti-CD3 antibodies of the present invention can be used to manufacture drugs for treating genetic diseases. The anti-CD3 antibodies of the present invention can be used to manufacture drugs for treating AIDS. The anti-CD3 antibodies of the present invention can be used to manufacture drugs for treating diabetes. The anti-CD3 antibody of the invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is conjugated to two folate and two pegylated molecules. In another example, a non-naturally encoded amino acid is site-specifically incorporated into the anti-CD3 Fab antibody and conjugation to two folate and two PEGylated molecules is via the non-natural amine group The side chain of the acid is realized.

The present disclosure provides an anti-CD3 antibody or antibody fragment or variant having one or more non-naturally encoded amino acids incorporated into the antibody or fragment or variant. The anti-CD3 antibodies or antibody fragments or variants may include, but are not limited to, Fv, Fc, Fab and (Fab')2, single chain Fv (scFv), Diabody, tribody, tetrabody, bifunctional hybrid antibody, CDR1, CDR2, CDR3, CDR combination, variable region, framework region, constant region, heavy chain, light chain, alternative scaffold non-antibody molecules, Bispecific antibodies, etc. In one embodiment, the anti-CD3 antibody or antibody fragment or variant is an anti-CD3 Fab antibody, fragment or variant.

The present disclosure provides bispecific antibody conjugates comprising anti-CD3 antibodies or antibody fragments. Disclosed herein are bispecific antibody conjugates comprising anti-CD3 Fab antibodies or antibody fragments incorporating one or more non-naturally encoded amino acids. The invention further discloses bispecific antibody conjugates 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 antibodies. The present invention discloses bispecific antibody conjugates 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 position-specifically Incorporated into anti-CD3 Fab antibodies. The invention discloses anti-CD3 Fab bispecific antibodies, which comprise: an antibody or an 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 linkers through the one or more linkers. The one or more small molecules are linked, and wherein the small molecule is one or more folates or one or more DUPA molecules or analogs or derivatives. The antibody or antibody fragment can be site-specifically linked to one or more folates or one or more DUPA molecules via one or more linkers. An antibody or antibody fragment may contain one or more unnatural amino acids. The antibody or antibody fragment may be linked to one or more folic acid or one or more DUPA molecules via one or more linkers, which link to one or more unnatural amino acids. Unnatural amino acids can be site-specifically incorporated into antibodies. The antibody or antibody fragment can be linked to one or more folic acid or DUPA molecules through one or more PEG molecules linked to one or more unnatural amino acids. The antibody or antibody fragment may alternatively be linked to one or more folic acid or one or more DUPA molecules via one or more linkers, which link to the natural amino acid. The antibody or antibody fragment may be an anti-CD3 Fab.

The invention discloses bispecific antibody conjugates, which comprise: anti-CD3 Fab; one or more folate molecules; and one or more linkers, wherein the anti-CD3 Fab is connected to the said one or more linkers through the one or more linkers. One or more folate molecules are attached. Anti-CD3 Fab may contain one or more unnatural amino acids. The one or more non-natural amino acids may replace the natural amino acids of the anti-CD3 Fab. The invention discloses bispecific antibody conjugates, which comprise: anti-CD3 Fab; one or more DUPA molecules; and one or more linkers, wherein the anti-CD3 Fab is connected to the said one or more linkers through the one or more linkers. One or more DUPA molecules are connected. The anti-CD3 Fab may comprise one or more unnatural amino acids. The one or more non-natural amino acids may replace the natural amino acids of the anti-CD3 Fab.

The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth exemplary embodiments that utilize the principles of the present invention, and the accompanying drawings.

CD25+: Positive activation of T cell markers

CD69+: positive for activation of T cell markers

_CH 1: Heavy chain constant region

C _L : light chain constant region

Fab: Antigen binding segment

Fab 1~Fab 14: Antigen binding segment 1~14

Fab 16: Antigen binding segment 16

Fab 17: Antigen binding segment 17

Fab 21: Antigen binding segment 21

Fab1-HK129: engineered heavy chain variant of anti-CD3 Fab

Fab1-HK129-pAF: engineered heavy chain pAF variant of anti-CD3 Fab

Fab1-HK129-LL157-pAF: engineered heavy chain-light chain (double) pAF variant of anti-CD3 Fab

CD3(HK129pAF): engineered anti-CD3 heavy chain pAF variant

CD3 (HK129pAF-LL157pAF): engineered heavy chain-light chain (double) pAF variant of anti-CD3 Fab

α CD3 Fab: Anti-CD3 Fab

G1~G6: treatment group

KB: cell line

OV-90: cell line

SKOV-3: cell line

hCD45+: human CD45 positive

hCD45+/mCD45+: human CD45/mouse CD45 positive

M1, M2: human macrophages

N: Nitrogen atom

O:Oxygen atom

PEG: polyethylene glycol

PEGx: x molecules of polyethylene glycol

SDS-PAGE: polyacrylamide gel electrophoresis

T1/2: half-life

TIL: tumor infiltrating lymphocytes

_VH : Heavy chain variable region

V _L : light chain variable region

Figure 1 depicts the binding of humanized anti-CD3 Fab to human peripheral blood mononuclear cells (PBMC). Binding of humanized anti-CD3 Fab expressed in HEK293 cells to human PBMC is shown (2 experiments). A total of 9 Fabs were tested by combining 3 vH and 3 vL chain sequences. The three Fabs containing the vL1.0 chain lost binding to human CD3 when combined with any vH chain.

Figures 2A-2F depict titrations of humanized anti-CD3 Fab binding to human and cynomolgus monkey PBMC. (Figure 2A), Fab 1 titration; (Figure 2B), Fab 2 titration; (Figure 2C), Fab 3 titration; (Figure 2D), Fab 4 titration; (Figure 2E), Fab 5 titration; and (Figure 2F), Fab 6 titration.

Figure 3 depicts rat pharmacokinetic (PK) analysis of anti-CD3 Fab1 molecules. Pegylation of humanized anti-CD3 Fab1 molecules increases plasma half-life (T1/2) in rats.

Figures 4A-4B depict the binding of Round 1 low affinity Fabs from HEK293 cells. Round 1 anti-CD3 Fab variants with reduced binding affinity for human (Fig. 4A) and cynomolgus monkey (Fig. 4B) CD3 produced by HEK293 cells are shown. Among the 35 new Fab variants tested in round 1, titration curves for 4 selected (Fas 7 to 10) reduced affinity Fabs as well as the control Fab1 are shown.

Figures 5A-5B depict binding of round 2 low affinity Fabs from HEK293 cells to human CD3. Round 2 anti-CD3 Fab variants with reduced binding affinity for human (Fig. 5A) and cynomolgus monkey (Fig. 5B) CD3 produced by HEK293 cells are shown. Of the 43 new Fab variants tested in Round 2 screening, titration curves are shown for 4 selected affinity-reducing Fabs as well as the parent control Fab1.

Figures 6A-6B depict binding of low affinity Fab-folate variants from E. coli cells to human CD3. Figure 6A shows the first round of binding, and Figure 6B shows the second round of binding of a low-affinity variant of the anti-CD3 Fab-HK129-folate molecule purified from E. coli cells to human CD3. Fab1-HK129-Folate (parental control) was included as a positive control.

Figures 7A-7B depict low affinity variants of anti-CD3 Fab-folate binding to cynomolgus CD3. Figure 7A shows the first round of binding, and Figure 7B shows the second round of binding of a low-affinity variant of the anti-CD3 Fab-HK129-folate molecule purified from E. coli cells to cynomolgus CD3. Fab1-HK129-Folate (parental control) was included as a positive control.

Figure 8 depicts cytotoxicity on SKOV-3 cells using human PBMC. In vitro cytotoxicity of 4 low-affinity variants of humanized anti-CD3 Fab-HK129-folate molecules produced from E. coli cells using human PBMC. Fab1-HK129-Folate (parental control) was included as a positive control.

Figure 9 depicts cytotoxicity against SKOV-3 cells using cynomolgus monkey PBMC. In vitro cytotoxicity of 4 low-affinity variants of the humanized anti-CD3 Fab-HK129-folate molecule produced from E. coli cells using cynomolgus monkey PBMC. Fab1-HK129-Folate (parental control) was included as a positive control.

Figures 10A-10B depict activation of T cells. Figure 10A shows activation of the T cell markers CD25 and CD69 by various low affinity anti-CD3 Fab-HK129-folate molecules in the absence of SKOV-3 cells. Figure 10(B) shows the activation of T cell markers CD25 and CD69 by various low affinity anti-CD3 Fab-HK129-folate molecules in the presence of SKOV-3 cells.

Figures 11A-11D depict the performance of various low affinity anti-CD3 Fab-HK129-folate variants in the absence (Figures 11A and 11C) or presence of SKOV-3 tumor cells (Figures 11B and 11D). In-vitro cytokine release of IFNγ (Figures 11A and 11B) and TNFα (Figures 11C and 11D).

Figures 12A-12F depict schematic representations of anti-CD3 Fab-folate bispecific conjugates and PEG linkers of the invention. Figure 12A is a representative example of folate-PEG ligand; Figure 12B is a representative example of folate-branched PEG ligand; Figure 12C-12D is a representative example of the conjugate of CD3-folate bispecific antibody and PEG Figure 12E is a representative graphical representation of a CD3-folate bispecific antibody with C-terminal PEGylation; and Figure 12F is a representative graphical representation of a CD3-folate bispecific antibody with C-terminal PEGylation by CD3-Fab cross-linking Representative illustration of specific antibodies.

Figures 13A-13D depict CD3 Fab-folate conjugates in the presence and absence of PEGylation. Figures 13A and 13B show SDS-PAGE gel electrophoresis analysis of single and double conjugated anti-CD3 Fab compositions; Figure 13C shows SDS-PAGE gel electrophoresis analysis of anti-CD3 Fab-folate C-terminal PEG conjugate; Figure 13D shows the in vitro cytotoxicity of anti-CD3 Fab-folate C-terminal PEG conjugate in KB, OV-90 and SKOV-3 cells.

Figure 14 depicts in vitro cytotoxicity and shows that anti-CD3 Fab-folate bispecific antibodies selectively kill FOLRα-expressing KB cells.

Figures 15A-15B depict in vitro cytotoxicity. Figure 15A shows that anti-CD3 Fab-folate bispecific antibody selectively kills FOLRα expressing SKOV3 cells in the presence of 20 nM folate, and Figure 15B shows selective killing in the presence of 50 nM folate.

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

Figure 17 depicts mouse pharmacokinetic studies in CD1 mice.

Figures 18A-18B depict anti-CD3 Fab-folate bispecific antibodies that selectively kill human M2 macrophages. Figure 18A shows the cytotoxicity of monofolate-containing anti-CD3 Fab-folate bispecific antibodies on macrophages in vitro. Arrows and numbers indicate fold differences in IC50 between M1 and M2 macrophages. The dashed lines represent Fab1-HK129-folate in M1 and M2, while the solid lines represent Fab1-HK129-5KPEG-folate in M1 and M2. Figure 18B shows the cytotoxicity of bifolate-containing anti-CD3 Fab-folate bispecific antibodies on macrophages in vitro. Arrows and numbers indicate fold differences in IC50 between M1 and M2 macrophages. The dashed line represents Fab1-HK129-LL157-bisfolate in M1 and M2, while the solid line represents Fab1-HK129-LL157-bisfolate-bis5KPEG in M1 and M2.

Figures 19A-19B depict 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.

Figures 20A-20B depict 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.

Figures 21A-21F depict anti-tumor efficacy in human cervical tumors derived from the KB cell line by multiple dose administration of a CD3-folate bispecific antibody (Figure 21A shows tumor growth, Figure 21B shows body heavy) and the expression of T cell activation markers CD25 (Fig. 21C), CD69 (Fig. 21D), CD45 (Fig. 21E) and CD3 (Fig. 21F).

Figures 22A-22B depict the anti-tumor efficacy of CD3-folate bispecific antibodies in human cervical tumors derived from the KB cell line; Figure 22A shows tumor growth and Figure 22B shows body weight.

Figures 23A-23B depict the anti-tumor efficacy of CD3-folate bispecific antibodies compared to carboplatin in human ovarian tumors derived from the OV-90 cell line; Figure 23A shows tumor growth and Figure 23B shows body weight.

It is to be understood that this invention is not limited to the specific methods, protocols, cell lines, constructs and reagents described herein, and may vary accordingly. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Definition:

As used in this disclosure and the appended claims, references without a specific number include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "anti-CD3 Fab" or "anti-CD3 Fab-folate antibody" is a reference to one or more such proteins and includes equivalents thereof known to those skilled in the art, and the like. The term "PEG-folate" or "folate-PEG" and the term "diPEG-bifolate" or "bifolate-diPEG" are used interchangeably herein.

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. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, only the preferred methods, devices, and materials are now described.

All publications and patents mentioned herein are hereby incorporated by reference to describe and disclose, for example, the constructs and methods described in the publications, which may be used in connection with the presently described invention. The publications discussed herein are provided solely with respect to their disclosures prior to the filing date of this application. Nothing contained in this disclosure shall be construed as an admission that the inventor is not entitled to precede this disclosure by reason of prior invention or for any other reason.

The term "substantially purified" refers to an anti-CD3 Fab antibody that may be substantially or essentially free of groups found in its naturally occurring environment that normally accompany or interact with the protein. Specifically, the naturally occurring environment is native cells or, in the case of recombinantly produced anti-CD3 antibodies, host cells. Anti-CD3 antibodies that can be substantially free of cellular material include protein preparations that have 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% contaminating protein. When anti-CD3 antibodies or variants thereof are recombinantly produced by host cells, the protein can be present at about 30%, about 25%, about 20%, about 15%, about 10%, or about the dry weight of the cells. Present in an amount of about 5%, about 4%, about 3%, about 2%, or about 1% or less. When an anti-CD3 antibody or a variant thereof is recombinantly produced by a host cell, the protein can be present at about 5 g/L, about 4 g/L, about 3 g/L, or about 2 g dry weight of the cells. /L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L or about 1mg/L or less present in the periplasm ( periplasm) and/or culture medium. Accordingly, a "substantially purified" anti-CD3 antibody as produced by the methods of the invention can have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55% , a purity level of at least about 60%, at least about 65%, at least about 70%, specifically, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, at least A purity level of about 95%, and at least a purity level of about 99% or greater, as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC and capillary electrophoresis.

"Recombinant host cell" or "host cell" refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transformation, transduction, f-mating or Other methods for generating recombinant host cells are known in the art. The exogenous polynucleotide can be maintained as a nonintegrated vector, such as a plasmid, or can be integrated into the host genome.

Antibodies are proteins that exhibit binding specificity for a specific antigen. Native antibodies are usually heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide linkages varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domains of the light chain are aligned with the first constant domain of the heavy chain, and the variable structure of the light chain The domains are aligned with the variable domains of the heavy chain. It is believed that specific amino acid residues form an interface between the variable domains of the light and heavy chains.

The term "variable" refers to the fact that certain portions of the variable domain vary widely in sequence between antibodies and are responsible for the binding specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed across the variable domains of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) in the variable domains of both light and heavy chains. The highly conserved parts of the variable domains are called framework regions (FR). The variable domains of the native heavy and light chains each contain four FR regions, mainly in a β-sheet configuration, connected by three CDRs that form a loop connecting the β-sheet structure, and in some cases form part of the β-sheet structure. The CDRs in each chain are tightly bound together by the FR region and, together with the CDRs in the other chain, contribute to the antigen binding of the antibody. The formation of the antigen binding site (see Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991) )).

The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions. Antibodies or immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), for example, IgG is divided into IgG1, IgG2, IgG3, and IgG4; IgA is divided into IgA1 and IgA2. The heavy chain constant regions corresponding to the 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 k and lambda, respectively. Among the various human immunoglobulin classes, only human IgGl, IgG2, IgG3 and IgM are known to activate complement. The immunoglobulin may be selected from IgG, IgA, IgD, IgE, IgM or fragments or modifications thereof.

In vivo, affinity maturation of antibodies is driven by antigenic selection of higher affinity antibody variants, which are primarily generated by somatic hypermutagenesis. "Repertoire shift" also often occurs, in which the dominant germline genes for secondary or tertiary reactions look different from the dominant germline genes for primary or secondary reactions.

The immune system's affinity maturation process can be replicated by introducing mutations into antibody genes in vitro and using affinity selection to isolate mutants with improved affinity. Such mutant antibodies can be displayed on the surface of filamentous phage or microorganisms such as E. coli, yeast, and the antibodies can be selected by their affinity for the antigen or by the kinetics of their dissociation from the antigen (off-rate). Hawkins et al., J. Mol. Biol. 226:889-896 (1992). CDR walking mutagenesis has been used to affinity mature human antibodies that bind the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) (Barbas III et al., PNAS (USA) 91 : 3809-3813 (1994); and Yang et al., J. Mol. Biol. 254: 392-403 (1995)); and anti-c-erbB-2 single chain Fv fragment (Schier et al., J. Mol. Biol. 263 :551567(1996)). Targeting the third hypervariable loop of HIV using antibody chain shuffling and CDR mutagenesis Affinity maturation of high-affinity human antibodies (Thompson et al., J. Mol. Biol. 256:77-88 (1996)). Balint and Larrick, Gene 137:109-118 (1993) describe computer-assisted oligodeoxyribonucleotide-directed scanning mutagenesis, whereby all CDRs of a variable region gene are searched simultaneously and exhaustively for improved mutations. body. αvβ3-specific humanized antibodies were affinity matured using an initial limited mutagenesis strategy in which each position of all six CDRs was mutated, followed by expression and selection of genes containing the highest affinity Combinatorial library of mutants (Wu et al., PNAS (USA) 95:6037-6-42 (1998)). Phage-displayed antibodies are reviewed in Chiswell and McCafferty TIBTECH 10:80-84 (1992); and Rader and Barbas III Current Opinion in Biotech. 8:503-508 (1997). Mutated antibodies having amino acid substitutions in the CDRs with increased affinity compared to the parent antibody are reported in each case.

"Affinity maturation" in the present invention refers to the process of enhancing the affinity of an antibody for its antigen. Methods for affinity maturation include, but are not limited to, computational screening methods and experimental methods.

"Antibody" in the present invention refers to a protein consisting of one or more polypeptides substantially encoded by all or part of antibody genes. Immunoglobulin genes include, but are not limited to, kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3 and IgG4), delta, epsilon and mu constant region genes, as well as numerous immunoglobulin variable region genes. Antibodies in the present invention are intended to include full-length antibodies and antibody fragments, and include naturally occurring or engineered antibodies (eg, variants) in any organism. The antibodies disclosed in the present invention can be human antibodies, humanized antibodies, engineered antibodies, non-human antibodies and/or chimeric antibodies. Humanized antibodies and methods for making them are well known in the art. (See, eg, U.S. Patent Nos. 5,821,337; 7,527,791; 6,982,321; 7,087,409; 5,766,886). Typically, a humanized antibody contains 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. Humanized antibodies may optionally comprise at least a portion of a human constant region. In one embodiment, for example, framework residues in a humanized antibody can be replaced by corresponding residues from a non-human antibody. Residues are substituted to restore or improve the specificity or affinity of the non-human antibody from which the CDR residues are derived. Chimeric antibodies may refer to antibodies produced by combining or joining two or more antibody genes that originally encode separate antibodies. For example, chimeric antibodies can be produced by combining or joining two or more antibody genes (or fragments thereof) from human, bovine, or murine species. In one embodiment, at least a portion of the antibody or antibody fragment may be derived from humans or cynomolgus monkeys, but is not limited thereto. In certain embodiments, the antibodies disclosed herein may have cross-species reactivity, for example, the antibodies may recognize human antigens and cyno antigens (eg, a human/cyno antibody).

"Antibody fragment" refers to any form of an antibody other than the full-length form. Antibody fragments of the invention include smaller components of antibodies present in full-length antibodies and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab and (Fab')2, single chain Fv (scFv), diabody, tribody, tetrabody, bifunctional hybrid antibody, CDR1, CDR2, CDR3, CDR Combinatorial, variable region, framework region, constant region, heavy chain, light chain, alternative scaffold non-antibody molecules, bispecific antibodies, etc. (Maynard and Georgiou, 2000, Annu. Rev. Biomed. Eng. 2: 339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9: 395-402). Statements and claims using the term "antibody" may specifically include "antibody fragments" unless expressly stated otherwise. In certain embodiments, "anti-CD3 antibody", "anti-CD3 Fab", "anti-CD3 Fab antibody" and "anti-CD3 Fab variant" are antibody fragments as defined herein.

"Computational screening method" as used herein refers to any method for designing one or more mutations in a protein, wherein the method uses computers to evaluate potential amino acid side chain substitutions relative to each other and /or the energy of the interaction with the rest of the protein.

The "full-length antibody" in the present invention refers to the structure that constitutes the natural biological form of the H and/or L chain of the antibody. In most mammals, including humans and mice, this form is a tetramer and Consists of two identical pairs of two immunoglobulin chains, each pair having a light chain and a heavy chain, each light chain containing the immunoglobulin domains VL and CL, and each heavy chain containing the immunoglobulin domain VH , Cγ1, Cγ2 and Cγ3. In each pair, the light and heavy chain variable regions (VL and VH) are jointly responsible for binding to the antigen, and the constant regions (CL, Cγ1, Cγ2 and Cγ3, especially Cγ2 and Cγ3) are responsible for the antibody effector functions. In some mammals, such as camels and llamas, full-length antibodies may consist of only two heavy chains, each containing the immunoglobulin domains VH, Cγ2, and Cγ3.

"Immunoglobulin (Ig)" in the present invention refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have a variety of structural forms, including, but not limited to, full-length antibodies, antibody fragments, and individual immunoglobulin domains, including, but not limited to, VH, Cγ1, Cγ2, Cγ3, VL, and CL.

The "immunoglobulin (Ig) domain" in the present invention refers to a protein domain consisting of a polypeptide substantially encoded by an immunoglobulin gene. Ig domains include, but are not limited to, VH, Cγ1, Cγ2, Cγ3, VL and CL.

As used herein, "variant 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 the native wild-type protein sequence, or another variant of the wild-type sequence. Typically, the starting sequence is referred to as the "parent" sequence, and may be a wild-type or variant sequence. For example, preferred embodiments of the present invention may utilize humanized parent sequences and perform computational analyzes thereon to generate variants.

The "variable region" of an antibody in the present invention refers to one or more polypeptides consisting of a VH immunoglobulin domain, a VL immunoglobulin domain, or a VH and VL immunoglobulin domain (including variants). The variable region may refer to such a polypeptide isolated as an Fv fragment, a scFv fragment, this region in the case of a larger antibody fragment, or this region in the case of a full-length antibody, or alternatively a non-antibody scaffold molecule.

With respect to the anti-CD3 Fab antibodies of the invention, the term "antigenically specific" or "specifically binds" refers to one or more expressions of the antigen of interest or binding partner. Anti-CD3 antibodies that epitopes bind but do not substantially recognize and bind to other molecules in a sample containing a mixed population of antigens.

As used herein, the term "bispecific anti-CD3 antibody" or "multispecific anti-CD3 antibody" refers to an anti-CD3 antibody that contains two or more antigen binding sites or binding partner binding sites, with the first binding site There is affinity for a first antigen or epitope and the second binding site has binding affinity for a second antigen or epitope that is different from the first antigen or epitope. In one embodiment, a bispecific anti-CD3 antibody has a binding site that binds CD3 and one or more binding sites with binding affinities for different antigens or epitopes.

As used herein, the term "epitope" refers to a location on an antigen or binding partner that is recognized by an anti-CD3 Fab antibody. If the antigen comprises a polypeptide, the epitope may be a linear or conformational amino acid sequence or shape. An epitope can also be any position on any type of antigen that allows an anti-CD3 antibody to bind to that antigen.

As used herein, "antigen-binding polypeptide" or "anti-CD3 Fab antibody" shall include at least those polypeptides and proteins that possess the biological activity of specifically binding to a specific binding partner. For example, antigens, as well as CD3 analogs, CD3 isoforms, CD3 mimetics, CD3 fragments, hybrid CD3 proteins, fusion proteins thereof, oligomers and polymers, homologs (homologues), glycosylation pattern variants and mutant proteins, regardless of their biological activity and further without regard to their methods of synthesis or manufacture, including but not limited to recombinant (whether from cDNA, genomic DNA, synthetic DNA or other forms Nucleic acid production), in vitro, in vivo, by microinjection of nucleic acid molecules, synthesis, transgenic and gene activation methods. Specific examples of anti-CD3 antibodies include, but are not limited to, antibody molecules, heavy chains, light chains, variable regions, CDRs, Fab, scFv, alternative scaffolds Alternative scaffold non-antibody molecules, ligands, receptors, peptides or any amino acid sequence that binds to an antigen.

Antigen-binding polypeptides include pharmaceutically acceptable salts and prodrugs, as well as prodrugs, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants and naturally occurring human antibodies of such salts. Stereoisomers of CD3 antibodies as well as agonist, mimetics and antagonist variants of naturally occurring human anti-CD3 antibodies and polypeptide fusions thereof. The term "antigen-binding polypeptide" encompasses fusions containing additional amino acids at the amine terminus, the carboxyl terminus, or both. Exemplary fusions include, but are not limited to, for example, a methionyl anti-CD3 antibody in which methionine is linked to the N-terminus of an anti-CD3 antibody produced by recombinant expression, fusions for purification purposes (including but not limited to polyhistidine or affinity epitopes), fusions for the purpose of linking anti-CD3 antibodies to other bioactive molecules, fusions to serum albumin-binding peptides, and fusions to serum proteins e.g. Fusion of serum albumin.

The term "antigen" or "binding partner" refers to a substance that is the target of the binding activity exhibited by an anti-CD3 antibody. Almost any substance can be an antigen or binding partner for anti-CD3 Fab antibodies.

Naturally produced antibodies (Ab) are tetrameric structures composed of two identical immunoglobulin (Ig) heavy chains and two identical light chains. The heavy and light chains of Ab are composed of different domains. Each light chain has one variable domain (VL) and one constant domain (CL), while each heavy chain has one variable domain (VH) and three constant domains (CH). Each domain, consisting of approximately 110 amino acid residues, is folded into a characteristic sandwich structure formed by two β-sheets stacked on top of each other, the immunoglobulin fold. The VL domains each have three complementarity determining regions (CDR1-3), while the VH domains each have up to three complementarity determining regions (CDR1-3), which are loops or loops connecting the beta strands at one end of the domain. The variable regions of both light and heavy chains generally contribute to antigen specificity, but the contribution of each chain to specificity is not necessarily equal. By using randomized CDR loops, antibody molecules have evolved to bind large numbers of molecules.

Functional substructures of Ab can be prepared by proteolysis and recombinant methods. They include Fab fragments containing the VH-CH1 domain of the heavy chain and the VL-CL1 domain of the light chain joined by a single interchain disulfide bond, and Fv fragments containing only VH and VL domains, the Fc portion contains the non-antigen binding region of the molecule. In some cases, 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 will specifically bind their antigens. (L. Masat et al., PNAS 91:893-896, 1994). It is sometimes found that isolated light or heavy chains also retain some antigen-binding activity (Ward et al., Nature 341, 554-546, 1989).

Another functional substructure is the single-chain Fv (scFv), which consists of the variable regions of an immunoglobulin heavy and light chain covalently linked by a peptide linker (S-z Hu et al., Cancer Research, 56, 3055-3061, 1996). These small (Mr 25,000 Da) proteins typically retain specificity and affinity for the antigen in a single polypeptide and can provide suitable building blocks for larger antigen-specific molecules. In many cases, the short half-life of scFvs in circulation limits their therapeutic utility.

Using part of the Ig VH domain as a template, a small protein scaffold called a "minibody" was designed (Pessi et al., Nature 362, 367-369, 1993). We identified mutants with high affinity for interleukin-6 (dissociation constant (Kd) of approx. 10-7M) minibody (Martin et al., EMBO J. 13, 5303-5309, 1994).

When camel serum was analyzed for IgG species, camels often lack variable light chain domains, suggesting that sufficient antibody specificity and affinity can be generated from the VH domain alone (three or four CDR loops). "Camelized" VH domains have been produced with high affinity and can produce high specificity by randomizing only the CDR3.

An alternative to "minibodies" is "diabodies." Diabodies are small bivalent and bispecific antibody fragments with two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). Diabodies are similar in size to Fab fragments. By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain and creates two antigen binding sites. These dimeric antibody fragments or "diabodies" are bivalent and bispecific. (See P. Holliger et al., PNAS 90:6444-6448, 1993).

CDR peptides and organic CDR mimetics have been prepared (Dougall et al., 1994, Trends Biotechnol. 12, 372-379). CDR peptides are short, usually cyclic peptides that correspond to the amino acid sequences of antibody CDR loops. CDR loops are responsible for antibody-antigen interactions. CDR peptides and organic CDR analogs have been shown to retain some binding affinity (Smyth and von Itzstein, J. Am. Chem. Soc. 116, 2725-2733, 1994). Mouse CDRs have been grafted onto the human Ig framework without loss of affinity (Jones et al., Nature 321, 522-525, 1986; Riechmann et al., 1988).

In humans, specific Abs are selected from large libraries and amplified (affinity maturation). These processes can be reproduced in vitro using combinatorial library technology. The successful display of Ab fragments on the phage surface made it possible to generate and screen a large number of CDR mutations (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) are produced through this technology. Combination techniques can be combined with Ab analogs (mimics).

A number of protein domains that may serve as protein scaffolds have been expressed as fusions with phage capsid proteins. Reviewed in Clackson and Wells, Trends Biotechnol. 12:173-184, 1994. Some of these protein domains have been used as scaffolds for displaying random peptide sequences, including 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 1:70-751994) and the IgG binding domain of Streptococcus (O'Neil et al., Techniques in Protein Chemistry V (edited by Crabb, L), pp. 517-524, Academic Press, San Diego, 1994). these The scaffold shows a single randomized ring or zone. Amystatin has been used as a presentation scaffold on filamentous bacteriophage M13 (McConnell and Hoess, J. Mol. Biol. 250:460-470, 1995).

The covalent attachment of the hydrophilic polymer polyethylene glycol (abbreviated as PEG) is beneficial for many bioactive molecules (including proteins, peptides, especially hydrophobic molecules) to increase water solubility, bioavailability, extend serum half-life, and extend Methods to treat half-life, modulate immunogenicity, modulate biological activity, or extend circulation time. PEG has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, nontoxicity, and nonimmunogenicity are important. To maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer(s) attached to the bioactive molecule must be high enough to confer the favorable properties typically associated with PEG polymer attachment, such as increased water solubility. and circulating half-life without adversely affecting the biological activity of the parent molecule.

PEG derivatives are often linked to bioactive molecules through reactive chemical functional groups such as lysine, cysteine and histidine residues, N-termini and carbohydrate moieties. Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play an important role in receptor binding and are necessary to maintain the biological activity of the molecule. As a result, indiscriminate attachment of polymer chains to these reactive sites on bioactive molecules often results in a significant reduction or even complete loss of the bioactivity of the polymer-modified molecule. (R. Clark et al., J. Biol. Chem., 271:21969-21977, 1996). In order to form conjugates with sufficient polymer molecular weight to confer the desired advantages on a target molecule, prior art methods often involve randomly attaching many polymer arms to the molecule, thereby increasing the biological activity of the parent molecule, reducing or even completely Risk of loss.

The location of the reaction that forms the site where the PEG derivative attaches to the protein is determined by the structure of the protein. Proteins (including enzymes) are composed of various alpha-amino acid sequences whose general structure is H2N--CHR--COOH. The α-amino part (H2N--) of an amino acid joins the carboxyl part (--COOH) of the adjacent amino acid to form an amide bond, which can be expressed as--(NH--CHR--CO)n --, where the subscript "n" can be To equal hundreds or thousands. The fragment represented by R may contain reactive sites for protein biological activity and for attachment of PEG derivatives.

For example, when the amino acid is lysine, a --NH2 moiety is present in the ε position and the α position. ε--NH2 can react freely under alkaline pH conditions. Much of the existing technology in the field of protein derivatization with PEG focuses on developing PEG derivatives to attach 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, these PEG derivatives all share the common limitation that they cannot be selectively installed on the numerous lysine residues present on the protein surface. In cases where the lysine residue is important for protein activity (e.g. present in the active site of an enzyme) or when the lysine residue plays a role in mediating interactions of the protein with other biomolecules (as in (as in the case of receptor binding sites), this may be a significant limitation.

A second, equally important complication with existing methods for protein PEGylation is that PEG derivatives may undergo undesirable side reactions with undesirable residues. Histidine contains a reactive imine moiety, structurally represented as --N(H)--, but many chemically reactive species that react with ε--NH2 can also react with --N(H)-- reaction. Similarly, the amino acid cysteine has a free sulfhydryl group on its side chain and is structurally represented as -SH. In some cases, PEG derivatives directed against the ε--NH2 group of lysine also react with cysteine, histidine, or other residues. This creates a complex heterogeneous mixture of PEG-derived bioactive molecules and creates the risk of disrupting the activity of the targeted bioactive molecule. It is desirable to develop PEG derivatives that allow the introduction of chemical functional groups at single positions within proteins, which thereby enable selective conjugation of one or more PEG polymers to bioactive molecules at specific, well-defined and predictable positions on the protein surface.

In addition to lysine residues, considerable effort in the art has been devoted to the development of activating PEG reagents targeting other amino acid side chains, including cysteine, histidine, and the N-terminus. See e.g. via U.S. Patent No. 6,610,281, incorporated by reference herein, and "Polyethylene Glycol and Derivatives for Advanced PEGylation," Nektar Molecular Engineering Catalog, pages 1-17, 2003 . Cysteine residues can be position-selectively introduced into the protein structure using site-directed mutagenesis and other techniques known in the art, and the resulting free thiol moieties can be combined with thiol-reactive functional groups reaction of PEG derivatives. However, this approach is complex because the introduction of free sulfhydryl groups complicates expression, folding, and stability of the resulting protein. Therefore, it would be desirable to have a method of introducing chemical functional groups into bioactive molecules that would enable selective conjugation of one or more PEG polymers to proteins while being compatible with sulfhydryl groups and other chemical functional groups commonly found in proteins. (i.e. not involved in undesirable side effects).

As noted in the art, a number of such derivatives have been developed for attachment to protein side chains, particularly the NH2 moiety on the lysine amino acid side chain and the cysteine side chain. -SH moiety, the synthesis and use of said derivatives have proven problematic. Some derivatives form unstable bonds with proteins that are susceptible to hydrolysis and thus break down, degrade, or are otherwise unstable in aqueous environments (eg, blood). Some derivatives form more stable bonds but undergo hydrolysis before forming the bond, meaning that the reactive groups on the PEG derivative may be deactivated before proteins can be attached. Some derivatives are slightly toxic and therefore less suitable for use in the body. Some derivatives react too slowly to be practical. Some derivatives cause loss of protein activity by attaching to the site responsible for protein activity. Some derivatives are not specific in the position at which they are to be attached, which may also lead to loss of the desired activity and lack of reproducibility of results. To overcome the challenges associated with modifying proteins with polyethylene glycol moieties, more stable PEG derivatives have been developed (e.g., U.S. Patent 6,602,498, which is incorporated by reference) or selected with thiol moieties on molecules and surfaces. Sexually reactive PEG derivatives (eg, U.S. Patent 6,610,281, which is incorporated herein by reference). There is a clear need in the art for PEG derivatives that are chemically inert in physiological environments until required and do not react selectively to form stable chemical bonds.

Recently, a completely new technology in protein science has been reported that promises to overcome many of the limitations associated with position-specific modification of proteins. Specifically, new components have been added to the prokaryotic Escherichia coli or E. coli (eg, L. Wang et al., Science 292:498-500, 2001) and the eukaryotic Saccharomyces cerevisiae or S. cerevisiae ) (eg, J. Chin et al., Science 301:964-7, 2003), which enables the incorporation of non-genetically encoded amino acids into proteins in vivo. Many novel amino acids with novel chemical, physical, or biological properties, including photoaffinity-labeled and photoisomerizable amino acids, keto-amino acids, and glycosylated amino acids, have been reported using this approach in response to The amber codon TAG is incorporated efficiently and with high fidelity into proteins in E. coli and yeast. See, for example, JW Chin et al., Journal of the American Chemical Society 124: 9026-9027, 2002; JW Chin and PG Schultz, (2002), ChemBioChem 11: 1135-1137; JW Chin et al., PNAS United States of America 99: 11020-11024, 2002; and L. Wang and PG Schultz, Chem. Comm., 1-10, 2002. These studies demonstrate that chemical functional groups such as ketone, alkynyl and azide moieties, which are not present in proteins, can be selectively and routinely introduced and are chemically inert to all functional groups present in 20 common genetically encoded amino acids. , and can be used to react efficiently and selectively to form stable covalent bonds.

The ability to incorporate non-genetically encoded amino acids into proteins allows the introduction of chemical functional groups, which can be naturally occurring functional groups such as lysine's ε-NH2, cysteine's sulfhydryl-SH, histamine The imine groups of acids, etc. provide valuable alternatives. Certain chemical functional groups are known to be inert toward functional groups found in 20 common genetically encoded amino acids, but they react cleanly and efficiently to form stable bonds. For example, it is known in the art to undergo a Huisgen [3+2] cycloaddition reaction of Azide and acetylene groups in the presence of catalytic amounts of copper under aqueous conditions. See, for example, Tornoe et al., (2002) Org. Chem. 67: 3057-3064; and Rostovtsev et al., (2002) Angew. Chem. Int. Ed. 41: 2596-2599. By introducing the azide moiety into the protein structure, for example, it is possible to incorporate chemically inert to amines, thiols, carboxylic acids, hydroxyl groups present in the protein but also able to react smoothly and efficiently with the acetylene moiety to form rings. Functional group of the addition product. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.

Various references disclose modification of polypeptides by polymer conjugation or glycosylation. The term "anti-CD3 antibody" or "antigen-binding polypeptide" refers to anti-CD3 antibodies as described above, as well as polypeptides that retain at least one biological activity of naturally occurring antibodies (including, but not limited to, activities other than antigen binding). Activities other than antigen binding include, but are not limited to, any one or more activities related to Fc. The term "anti-CD3 antibody" or "antigen-binding polypeptide" includes, but is not limited to, polypeptides conjugated to polymers such as PEG, and may contain cysteine, lysine, N- or C-terminal amino acids, or other residues one or more additional derivatizations. Additionally, the anti-CD3 antibody may comprise a linker, polymer or biologically active molecule, wherein the amino acid conjugated to the linker, polymer or biologically active molecule may be a non-natural amino acid according to the present invention or may utilize a linker, polymer or biologically active molecule known in the art. The technology conjugates naturally encoded amino acids, such as lysine or cysteine. US Patent No. 4,904,584 discloses pegylated lysine-depleted polypeptides in which at least one lysine residue has been deleted or replaced by any other amino acid residue. WO 99/67291 discloses a method of conjugating a protein to PEG in which at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein. WO 99/03887 discloses pegylated variants of polypeptides belonging to the growth hormone superfamily in which cysteine residues have been replaced by non-essential amino acid residues located in designated regions of the polypeptide. WO 00/26354 discloses a method for generating glycosylated polypeptide variants with reduced allergenicity, said variants comprising at least one additional glycosylation position compared to the corresponding parent polypeptide.

The term "antigen-binding polypeptide" also includes glycosylated anti-CD3 antibodies, such as, but not limited to, polypeptides that are glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered biologically active variants of anti-CD3 antibodies. Additionally, splice variants were included. The term "antigen-binding polypeptide" also includes any one or more anti-CD3 antibodies or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand or other biological material of any type. Anti-CD3 antibody heterodimers, homodimers, heteromultimers or homomultimers (chemically linked or expressed as fusion proteins) of biologically active molecules, and containing, for example, specific deletions or other modifications Peptide analogues that retain biological activity.

In one embodiment, the antigen-binding polypeptide further includes additions, substitutions, or deletions that modulate the biological activity of the anti-CD3 antibody. For example, additions, substitutions or deletions may modulate one or more properties or activities of the anti-CD3 antibody, including but not limited to modulating affinity for the antigen, modulating (including but not limited to increasing or decreasing) antigen conformation or other secondary or tertiary or changes in quaternary structure, stabilizes antigen conformation or other secondary, tertiary or quaternary structure changes, induces or causes changes in antigen conformation or other secondary, tertiary or quaternary structure, regulates circulation half-life, regulates therapeutic half-life , modulate the stability of the polypeptide, adjust dosage, modulate release or bioavailability, facilitate purification, or improve or change a specific route of administration. Similarly, antigen-binding polypeptides may comprise protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to FLAG or poly-His) or other affinity-based sequences (including but not limited to FLAG, poly-His, GST etc.) or linking molecules (including but not limited to biotin), which can improve the detection (including but not limited to GFP), purification or other properties of the polypeptide.

The term "antigen-binding polypeptide" also encompasses linked anti-CD3 antibody homodimers, heterodimers, homomultimers and heteromultimers, including, but not limited to, via non-naturally encoded amino acid sides. The chains are linked directly to the same or different non-naturally encoded amino acid side chains, to naturally encoded amino acid side chains (as a fusion), or indirectly via a linker. Exemplary linkers include, but are not limited to, small organic compounds, water-soluble polymers of various lengths such as polyethylene glycol or polydextran, or polypeptides of various lengths.

Those skilled in the art will appreciate that amino acid positions corresponding to positions in a particular antigen-binding polypeptide sequence can be readily identified in an antigen-binding polypeptide, or fragments of related antigen-binding polypeptides, or the like. For example, sequence alignment programs such as BLAST can be used to align and identify specific positions in proteins that correspond to positions in related sequences.

The term "antigen-binding polypeptide" encompasses antigen-binding polypeptides containing one or more amino acid substitutions, additions or deletions. The antigen-binding polypeptides of the present invention may comprise modifications of one or more natural amino acids in combination with modifications of one or more non-natural amino acids. Exemplary substitutions of various amino acid positions in naturally occurring anti-CD3 antibody polypeptides have been described, including, but not limited to, substitutions that modulate one or more biological activities of the antigen-binding polypeptide, such as, but not limited to, enhancing agonist activity, increasing the polypeptide's solubility, conversion of the polypeptide into an antagonist, etc., and these are encompassed by the term "anti-CD3 antibody".

"Non-naturally encoded amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolidine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally encoded amino acid" are "non-natural amino acid", "non-natural amino acid", "non-naturally occurring amino acid", "atypical amino acid""non-canonical amino acid" and its various hyphenated and unhyphenated forms. The term "non-naturally encoded amino acids" also includes, but is not limited to, modifications by naturally encoded amino acids including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine (e.g. Amino acids that appear due to post-translational modification), but they themselves cannot be naturally incorporated into the growing polypeptide chain through the translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N -acetylglucosaminyl-L-serine, N -acetylglucosaminyl-L-threonine, and O-phosphotyramine acid. In one embodiment, the non-natural amino acid contains a sugar moiety. Examples of such amino acids include N -acetyl-L-glucosamine-L-serine, N -acetyl-L-galactosamine-L-serine, N -acetyl-L-glucosamine-L-serine -L-glucosamine-L-threonine, N -acetyl-L-glucosamine-L-aspartic acid, and O-mannosamine-L-serine. Examples of such amino acids also include those in which the naturally occurring N- or O-bonds between the amino acid and the sugar are replaced by covalent bonds not commonly found in nature, including but not limited to alkenes, oximes, thioethers , amide, etc. Examples of such amino acids also include sugars uncommon in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxy-galactose, and the like. Specific examples of non-natural amino acids include, but are not limited to, p -acetyl-L-phenylalanine, p -propargyloxyphenylalanine, O-methyl- L-tyrosine ( o -methyl-L-tyrosine), L-3-(2-naphthyl)alanine (L-3-(2-naphthyl)alanine), 3-methyl-phenylalanine (3- methyl-phenylalanine), O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, Tri-O-acetyl-GlcNAc-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L- Phenylalanine (isopropyl-L-phenylalanine), p -azido-L-phenylalanine, p-acyl-L-phenylalanine, p- acyl-L-phenylalanine p -benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodine- Phenylalanine ( p -iodo-phenylalanine), p -bromophenylalanine, p -amino-L-phenylalanine and isopropyl-L-phenylalanine -phenylalanine) etc.

"Amine terminal modification group" refers to any molecule that can be attached to the amine terminus of a polypeptide. Similarly, "carboxy-terminal modifying group" refers to any molecule that can be attached to the carboxyl terminus of a polypeptide. Terminal modifying 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.

The terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemical "Chemically reactive moiety" is used in the art, and in this invention refers to a unique definable part or unit of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to refer to portions of a molecule that perform certain functions or activities and are reactive with other molecules.

The term "linkage", "linkage" or "linker" as used in the present invention refers to a group or bond usually formed as a result of a chemical reaction, and is usually a covalent bond. A hydrolytically stable bond means that the bond is substantially stable in water and does not react with water at available pH values. should, including but not limited to, for an extended period of time under physiological conditions, or even indefinitely. A hydrolytically unstable or degradable bond means that the bond is degradable in water or aqueous solutions, including, for example, blood. An enzymatically labile or degradable linkage means that the linkage can be degraded by one or more enzymes. As is understood in the art, PEG and related polymers may include degradable linkages in the polymer backbone or in linking groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with alcohol groups on the bioactive agent are typically hydrolyzed under physiological conditions to release the agent. Other hydrolytically degradable bonds include, but are not limited to: carbonate bonds; imine bonds resulting from the reaction of amines and aldehydes; phosphate ester bonds formed by reacting alcohols with phosphate groups; Hydrazone bond; acetal bond as the reaction product of aldehyde and alcohol; orthoester bond as the reaction product of formate and alcohol; and carboxyl group of the peptide through amine group (including but not limited to at the terminus of polymer such as PEG) peptide bonds formed; and oligonucleotide bonds formed by phosphoramidite groups (including, but not limited to, at polymer termini) and the 5' hydroxyl group of the oligonucleotide. Branched linkers can be used in the antigen-binding polypeptides of the invention.

When used in the present invention, the terms "biologically active molecule", "biologically active moiety" or "biologically active agent" refer to substances that affect biological systems, pathways, Any substance that has any physical or biochemical properties of a molecule or interaction, including but not limited to viruses, bacteria, phages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, bioactive molecules include, but are not limited to, molecules intended for use in the diagnosis, cure, alleviation, treatment or prevention of disease in humans or other animals, or to otherwise enhance the physical or mental health of humans or animals. any substance. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles . Types of bioactive agents suitable for use in the present invention include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, drugs, anxiolytics, hormones, growth factors, steroid preparations, toxins of microbial origin, etc.

The anti-CD3 Fab-folate antibodies of the invention can be conjugated to molecules such as PEG to improve in vivo delivery and pharmacokinetic properties. Leong et al described position-specific pegylation of the Fab' fragment of an anti-IL-8 antibody, which had reduced clearance compared with the non-PEGylated form and little or no loss of antigen-binding activity ( Leong, S.R. et al., (2001) Cytokine 16:106-119).

Many different cleavable linkers are known to those skilled in the art. See U.S. Patent Nos. 4,618,492; 4,542,2254,625,014. Mechanisms for release of agents from these linker groups include, for example, irradiation of photolabile bonds and acid-catalyzed hydrolysis. For example, US Patent No. 4,671,958 includes a description of immunoconjugates containing linkers that are cleaved at a target site in vivo by proteolytic enzymes of the patient's complement system. The length of the linker can be predetermined or selected based on the desired spatial relationship between the anti-CD3 antibody and the molecule to which it is linked. Given that numerous methods have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other reagents to antibodies, one skilled in the art will be able to determine appropriate methods for attaching a given reagent to an anti-CD3 antibody or other peptides.

"Bifunctional polymer" refers to a polymer containing two discrete functional groups capable of specifically reacting with other moieties, including but not limited to pendant amino acid groups, to form covalent or non-covalent bonds. Bifunctional linkers having one functional group that reacts with a group on a particular biologically active component and another functional group that reacts with a group on a second biological component can be used to form a linker that includes a first biologically active component, a bifunctional linker, and Conjugate of a second biologically active component. Many programs and linker molecules are known for attaching various compounds to peptides. See, for example, European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784, 4,680,338, 4,569,789, and 4,589,071, which are incorporated herein by reference. "Polyfunctional polymer" refers to a polymer containing two or more discrete functional groups capable of specifically reacting with other moieties, including but not limited to pendant amino acid groups, to form covalent or non-covalent key. Bifunctional polymers or polyfunctional polymers can be any desired component sublength or molecular weight, and may be selected to provide a specific desired spacing or conformation between one of the molecules linked to the anti-CD3 antibody.

As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. Attachment of water-soluble polymers to anti-CD3 antibodies can result in changes including, but not limited to, increased or modulated serum half-life, or increased or modulated therapeutic half-life relative to the unmodified form, modulated immunogenicity, modulated physical association Properties such as aggregation and multimer formation, altered receptor binding, and altered receptor dimerization or multimerization. The water-soluble polymer may or may not have biological activity of its own and may be used as a linker to attach anti-CD3 antibodies to other substances, including but not limited to one or more anti-CD3 antibodies, or one or more Bioactive molecules. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, its mono-C10 alkoxy or aryloxy derivatives (described in U.S. Patent No. 5,252,714, which is incorporated by reference) , monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, Dextran derivatives (including dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymers, polyoxyethylenated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, polysaccharides, cellulose and cellulose Derivatives (including but not limited to methylcellulose and carboxymethylcellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives Materials, polyvinyl ether and α-β-poly[[2-hydroxyethyl)-DL-aspartic acid, etc., or their mixtures. Examples of such water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

As used herein, the term "polyalkylene glycol or poly(alkene glycol)" refers to polyethylene glycol, polypropylene glycol, polybutylene glycol and their derivatives. The term "polyalkylene glycol or poly(alkene glycol)" encompasses both linear and branched polymers and has an average molecular weight between 0.1 kDa and 100 kDa. In one embodiment, "polyalkylene glycol" may be about 5K to 50K or between 5K to 50K. For example, other exemplary embodiments are listed in commercial supplier catalogs, such as Shearwater Corporation's catalog "Polyethylene glycols and polyethylene glycols for biomedical applications." Derivatives (Polyethylene Glycol and Derivatives for Biomedical Applications)” (2001). As used in the present invention, polyethylene glycols having molecular weights of 5 kDa, 10 kDa, 20 kDa, etc. are referred to in the present invention as "5K PEG", "10K PEG", "20K PEG, etc." respectively.

As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulating half-life of a modified anti-CD3 antibody relative to its unmodified form. Serum half-life was determined by taking blood samples at different time points after administration of anti-CD3 antibodies and determining the concentration of this molecule in each sample. The correlation of serum concentration with time allows calculation of serum half-life. An increase in serum half-life of at least about twofold is expected, but smaller increases may be useful, for example, if they enable a satisfactory dosing regimen or avoid toxic effects. In one embodiment, the increase is at least about three times, at least about five times, at least about ten times, at least about fifteen times, at least about twenty times, at least about twenty-five times, at least about thirty times, At least about forty times or at least about fifty times or more.

As used herein, the term "modulated therapeutic half-life" refers to a therapeutically effective amount of an anti-CD3 antibody or an anti-CD3 antibody containing a modified bioactive molecule that is positive or negative relative to the half-life of its unmodified form. negative change. Therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life ideally enables a specific beneficial dosing regimen, a specific beneficial total dose, or the avoidance of undesirable effects. In one embodiment, the increased therapeutic half-life is due to an increase in potency, an increase or decrease in binding of the modified molecule to its target, or an increase or decrease in another parameter or mechanism of action of the non-modified molecule.

When applied to a nucleic acid or protein, the term "isolated" means that the nucleic acid or protein is substantially free of other cellular components with which it is associated in its native state. It can be in a homogeneous state. Isolated materials may be in a dry or semi-dry state, or in solution, including but not limited to aqueous solutions. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the main substance present in the formulation, is substantially purified. In particular, isolated genes are flanked by genes and encode proteins other than the gene of interest. Open reading frame segregation in white matter. The term "purified" means that the nucleic acid or protein produces essentially a band in an electrophoresis gel. In particular, this means that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure or higher.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and their polymers in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise expressly limited, the term also refers to oligonucleotide analogs, which include PNA (peptidonucleic acid, peptide nucleic acid), DNA analogs (phosphorothioate) used in antisense technology ester, aminophosphate, etc.). Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequences explicitly stated. Specifically, degenerate codon replacement can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and/or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19: 5081, 1991; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8: 91-98, 1994 ).

The terms "polypeptide", "peptide" and "protein" are used interchangeably in the present invention and refer to a polymer of amino acid residues. That is, a description of a polypeptide applies equally well to a description of a peptide as to 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 non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked by covalent peptide bonds.

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 20 common amino acids (alanine, arginine, aspartate, aspartic acid, hemi- Cystine, glutamine, glutamic acid, glycine, histine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) as well as pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., alpha carbon bonded to hydrogen, carboxyl, amine, and R groups), such as homoserine, norleucine , methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid.

Amino acids may be referred to herein by their well-known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. Likewise, nucleotides can be referred to by their recognized one-letter codes.

"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 those nucleic acids that encode the same or essentially the same amino acid sequence, or essentially the same sequence when the nucleic acid does not encode an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at each position where alanine is designated by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide of the invention also describes each possible silent variation of the nucleic acid. One skilled in the art will recognize that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce function. Same molecule. Thus, every silent variation in the nucleic acid encoding a polypeptide is implicit in every such sequence.

With regard to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions or additions to nucleic acid, peptide, polypeptide or protein sequences alter, add or delete individual amino acids or small portions of amino acids in the coding sequence. are "conservatively modified variants" in which the change results in the replacement of an amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids. These conservatively modified variants are complementary to, and do not exclude, the polymorphic variants, interspecies homologs and alleles of the present invention. Homologs and alleles.

The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Aspartic acid (N), glutamic acid (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 (WH Freeman &Co.; 2nd ed. (December 1993))) ).

In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are identical. If sequences have a certain percentage of identical amino acids when compared over a comparison window or designated region and aligned for maximum correspondence using one of the following sequence comparison algorithms or as measured by manual alignment and visual inspection residues or nucleotides (i.e., about 60% identical, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identical within a specified region ), the sequences are "substantially identical". This definition also refers to the complement of test sequences. Identity may exist over a region of at least about 50 amino acids or nucleotides in length, or over a region of 75 to 100 amino acids or nucleotides in length, or when not specified, over the entire sequence or on polynucleotides or polypeptides.

For sequence comparison, one sequence is usually used as a reference sequence, which is compared to a test sequence. When using sequence comparison algorithms, the test sequence and reference sequence must be entered into the computer. Specify subsequence coordinates if necessary, and specify sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identities of the test sequence relative to the reference sequence based on the program parameters.

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

As used herein, the term "effective amount" refers to an amount of a (modified) non-natural amino acid polypeptide administered that will alleviate one or more symptoms of the disease, condition or disorder being treated to a degree. Compositions containing the (modified) non-natural amino acid polypeptides of the invention may be administered for preventive, potentiative and/or therapeutic treatment.

The term "enhancement" means increasing or prolonging the potency or duration of a desired effect. Thus, with respect to enhancing the effects of a therapeutic agent, the term "enhancement" refers to the ability to increase or prolong in potency or duration the effects of other therapeutic agents on the system. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system. When administered to a patient, the effective amount used will depend on the severity and duration of the disease, disorder, or condition, previous treatments, the patient's medical condition and response to the drug, and the judgment of the attending physician.

As used herein, the term "modified" refers to the presence of post-translational modifications on a polypeptide. The form "(modified)" terminology means that the polypeptide in question is optionally modified, that is, the polypeptide in question may or may not be modified.

The terms "post-translationally modified" and "modified" refer to any modification of a natural or non-natural amino acid that occurs after the amino acid has been incorporated into a polypeptide chain. By way of example only, the terms encompass co-translational in vivo modifications, post-translational in vivo modifications and post-translational in vivo modifications.

In preventive or therapeutic applications, a composition containing a (modified) non-natural amino acid polypeptide is administered in an amount sufficient to cure or at least partially prevent the symptoms of the disease, disorder or condition to a patient already suffering from the disease, disorder or condition. A patient with a disease, condition or disorder. Such amounts are defined as "prophylactically effective amounts" or "therapeutically effective amounts" and will depend on the severity and duration of the disease, disorder, or condition, prior treatments, the patient's health and response to the medications, and the discretion of the attending physician. judge. It is considered well within the skill of the art to determine such therapeutically effective amounts by routine experimentation (eg, dose escalation clinical trials).

The term "treatment" is used to refer to preventive and/or therapeutic treatment.

Unless otherwise stated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill in the art were employed.

More specific implementations are proposed below according to the present invention.

introduction:

The inventors have developed bispecific antibodies containing biologically active molecules or agents in the absence or presence of water-soluble polymer molecules. In certain embodiments, the invention provides bispecific antibodies comprising anti-CD3 antibodies, fragments or variants comprising one or more folate molecules in the absence or presence of one or more PEG molecules. One or more PEG molecules may be single or double PEG, for example, single or double 5K PEG, 10K PEG, 20K PEG or larger. One or more PEG molecules can be linear or branched. Anti-CD3 antibodies, fragments or variants include ones that have been engineered to have one or more non-naturally encoded amino acids at any suitable position in the heavy or light chain amino acid sequence, such as p-acetyl-phenylalanine ( pAF) anti-CD3 Fab antibody, the positions include but are not limited to positions 114, 115, 129 or 160 (according to Kabat numbering) in the heavy chain of the Fab, and positions 157, 172, 205 in the light chain (according to Kabat number). Bispecific antibodies can comprise one or more non-naturally encoded amino acids such as p-acetyl- Anti-CD3 Fab of phenylalanine (pAF). Bispecific antibodies can be composed of anti-CD3 Fabs that have been engineered to have a covalent linkage between the heavy chain (K129; Kabat numbering) and the light chain (L157; Kabat numbering) of the Fab. No.) with one or more non-naturally encoded amino acids, such as p-acetyl-phenylalanine (pAF). In some embodiments of the invention, proprietary oxime chemistry can be used to conjugate or link one or more PEG molecules to one or more folate molecules that are incorporated into the anti-CD3 antibody. Conjugation with an unnatural amino acid (eg, pAF) results in, for example, one or two PEG and/or folic acid molecules stably conjugated to the anti-CD3 Fab. The addition of one or more PEG molecules (e.g., 5K, 10K, 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. Pro-tumorigenic macrophages (M2) and MDSC cells were observed to be preferentially depleted by the pegylated anti-CD3 Fab-folate compositions disclosed herein. The results suggest that improved pharmacokinetic properties will require less frequent dosing and may avoid the need for administration with an infusion pump. As used herein, the terms "PEG-folate" or "folate-PEG" and "diPEG-bifolate" or "bifolate-diPEG" are used interchangeably.

Antibodies, antibody fragments and variants thereof:

The antibodies or antibody fragments or variants of the invention may be human, humanized, engineered, non-human and/or chimeric antibodies or antibody fragments. The antibodies or antibody fragments or variants provided by the invention may comprise two or more amino acid sequences. The first amino acid sequence can comprise a first antibody chain and the second amino acid sequence can comprise a second antibody chain. The first antibody chain can comprise a first amino acid sequence and the second antibody chain can comprise a second amino acid sequence. A chain of an antibody may refer to an antibody heavy chain, an antibody light chain, or a combination of a region or all of an antibody heavy chain and a region or all of an antibody light chain. As non-limiting examples, the invention provides antibodies comprising a heavy chain, or fragments or variants thereof, and a light chain, or fragments or variants thereof. Two amino acid sequences of an antibody, including two antibody chains, can be connected by one or more disulfide bonds, chemical linkers, peptide linkers, or combinations thereof. Chemical linkers include linkers via unnatural amino acids. Chemical linkers include linkers via one or more unnatural amino acids. Chemical linkers may include chemical Chemical conjugate. Peptide linkers include any amino acid sequence that joins two amino acid sequences. The peptide linker may comprise 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more Many, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. The peptide linker can be part of any antibody, including domains of the antibody, such as variable domains, CH1, CH2, CH3 and/or CL domains. In one embodiment, the heavy and light chains are linked, for example, via a peptide linker. In some cases, the heavy and light chains are linked, for example, by one or more disulfide bonds.

The antibodies, antibody fragments, and antibody variants of the present disclosure can interact with or engage antigens on effector cells. Effector cells may 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, red blood cells, platelets, neutrophils Granulocytes, monocytes, eosinophils, basophils, mast cells, NK cells, B cells or T cells. In one embodiment, the immune cells may be T cells, such as cytotoxic T cells or natural killer T cells. Antibodies or antibody fragments can interact with receptors on T cells such as, but not limited to, T cell receptors (TCR). TCR may include TCRα, TCRβ, TCRγ and/or TCRδ or TCRζ. The antibodies or antibody fragments disclosed in the present invention can bind to receptors on lymphocytes, dendritic cells, B cells, macrophages, monocytes, neutrophils and/or NK cells. The antibodies or antibody fragments disclosed herein can bind to cell surface receptors. The antibodies or antibody fragments disclosed herein can bind to folate receptors. The antibodies or antibody fragments disclosed herein can be conjugated to T cell surface antigens such as, but not limited to, 2-[3-(1,3-dicarboxypropyl)-ureido]glutaric acid (2-[3-( 1,3-dicarboxy propyl)-ureido]pentanedioic acid (DUPA for short) 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 by reference in its entirety.

In certain embodiments, the antibodies or antibody fragments disclosed herein are anti-CD3 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-CD3 antibodies or antibody fragments or variants disclosed herein can be humanized. Anti-CD3 antibodies or antibody fragments or variants disclosed herein include, but are not limited to, CD3 analogs, isomers, analogs, fragments or hybrids. Anti-CD3 antibodies or antibody fragments or variants of the invention include, but are not limited to, Fv, Fc, Fab and (Fab')2, single chain Fv (scFv), diabody, tribody, tetrabody, bifunctional hybrid Hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. The anti-CD3 antibodies or antibody fragments or variants of the invention comprise the sequences of SEQ. ID. NO: 1 to 62. The antibody, fragment or variant of the invention may be an anti-CD3 Fab antibody, fragment or variant. Antibodies, fragments or variants of the invention may comprise one or more anti-CD3 Fabs. An antibody, fragment or variant of the invention may comprise two anti-CD3 Fabs. In certain embodiments, the anti-CD3 antibody comprises a heavy chain and/or light chain amino acid sequence selected from the sequence of SEQ. ID. NO: 1 to 62. In certain embodiments, an anti-CD3 antibody consists of a heavy chain and/or light chain amino acid sequence selected from the sequence of SEQ. ID. NO: 1 to 62. In certain embodiments, the anti-CD3 antibody comprises SEQ. ,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 , the heavy chain amino acid sequence of any one of 50, 50, 51, 52, 53, 54, 55, 56 and 57; and SEQ.ID.NO: 7, 8, 9, 18, 19, 20, The light chain amino acid sequence of any of 39, 58, 59, 60, 61 and 62.

The invention also discloses anti-CD3 bispecific antibodies comprising unnatural amino acids. In certain embodiments, anti-CD3 bispecific antibodies or antibody fragments or variants include, but are not limited to, Fv, Fc, Fab and (Fab')2, single chain Fv (scFv), diabody, tribody, Tetrabody antibodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, bispecific antibodies, etc. In one embodiment, an anti-CD3 bispecific antibody or Antibody fragments or variants are anti-CD3 Fab bispecific antibodies, fragments or variants that contain one or more non-naturally encoded amino acids. Anti-CD3 Fab bispecific antibodies or antibody fragments or variants of the invention may comprise one or more sequences of SEQ. ID. NO: 1 to 62. The bispecific antibody, fragment or variant of the invention may be an anti-CD3 Fab antibody, fragment or variant. The anti-CD3 bispecific antibody may comprise a heavy chain and/or light chain amino acid sequence selected from the sequence of SEQ. ID. NO: 1 to 62. In one embodiment, the anti-CD3 antibody consists of a heavy chain and/or light chain amino acid sequence selected from the sequence of SEQ. ID. NO: 1 to 62. In certain embodiments, the anti-CD3 bispecific antibody comprises SEQ. ID. NO: 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, The heavy chain amino acid sequence of any one of 48, 49, 50, 50, 51, 52, 53, 54, 55, 56 and 57; and SEQ.ID.NO: 7, 8, 9, 18, 19 , the light chain amino acid sequence of any one of 20, 39, 58, 59, 60, 61 and 62. In one embodiment, the bispecific antibodies, fragments or variants disclosed herein specifically bind CD3. Bispecific antibodies, fragments or variants can have cross-species reactivity. Bispecific antibodies, fragments or variants can react cross-species with human and monkey antigens. In one embodiment, the antibody comprises cross-species reactive CDRs. Bispecific antibodies can be humanized antibodies.

Table 1: Novel amino acid sequences of humanized anti-CD3 variants with and without HC-DKTHT extension, where the positions of the non-natural amino acid pAF incorporation are underlined in the heavy and light chains. Also disclosed are all sequences in the table below where pAF is replaced by any other non-natural amino acid.

Unnatural amino acids:

The invention provides anti-CD3 antibodies, antibody fragments or variants comprising at least one non-naturally encoded amino acid. Introducing at least one non-naturally encoded amino acid into an anti-CD3 antibody may allow the application of conjugation chemistries that engage in specific chemical reactions with one or more non-naturally encoded amino acids without interacting with the 20 amines that are typically present Acid reaction.

In some embodiments of the present disclosure are anti-CD3 antibodies comprising one or more non-naturally encoded amino acids. The one or more non-natural amino acids may be encoded by codons that do not encode one of the 20 natural amino acids. One or more unnatural amino acids may be encoded by a nonsense codon (stop codon). The stop codon can be an amber codon. Amber codons can contain UAG sequence. The stop codon may be an ocher codon. Ocher codons may contain UAA sequences. The stop codon can be an opal or umber codon. Opal or umber codons may contain UGA sequences. One or more unnatural amino acids may be encoded by four-base codons.

The one or more unnatural amino acids may include, but are not limited to, p-azidophenylalanine (pAz), p-benzophenylalanine (pBpF), p-propargyloxyphenylalanine (pPrF), p-iodoamphetamine acid (pIF), p-cyanophenylalanine (pCNF), p-carboxymethylphenylalanine (pCmF), 3-(2-naphthyl)alanine (NapA), p-boranophenylalanine (pBoF), o-nitro Phenylalanine (oNiF), (8-hydroxyquinolin-3-yl)alanine (HQA) and (2,2'-bipyridin-5-yl)alanine (BipyA). The one or more unnatural amino acids may be β-amino acids (β3 and β2), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives , ring-substituted phenylalanine and tyrosine acid derivatives, linear core amino acids, diamino acids, D-amino acids, N-methylamino acids or combinations thereof. Non-natural amino acids may also include, but are not limited to: 1) Various substituted tyrosine and phenylalanine analogs, such as O-methyl-L-tyrosine, p-amino-L-phenylalanine, 3-nitrate -L-tyrosine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine and p-isopropyl-L-phenylalanine; 2) having an aryl azide that can be photo-crosslinked Amino acids with compounds and benzophenone groups; 3) Amino acids with unique chemical reactivity, including acetyl-L-phenylalanine, m-acetyl-L-phenylalanine, O-allyl -L-tyrosine, O-(2-propynyl)-L-tyrosine, p-ethylthiocarbonyl-L-phenylalanine and p-(3-oxobutyl)-L-phenylalanine; 4 ) Amino acids containing heavy atoms used for phasing in X-ray crystallography, including p-iodo-L-phenylalanine and p-bromo-L-phenylalanine; 5) Redox active amino acid dihydroxy-L-amphetamine Acid; 6) Glycosylated amino acids, including b-N-acetylglucosamine-O-serine and a-N-acetylgalactosamine-O-threonine; 7) With naphthyl and dansulfonyl groups and fluorescent amino acids with 7-aminocoumarin side chains; 8) Photolyzable and photoisomerizable amino acids with azobenzene and nitrobenzyl Cys, Ser and Tyr side chains; 9 ) the phosphotyrosine mimetic p-carboxymethyl-L-phenylalanine; 10) the glutamine homologue homoglutamic acid; and 11) 2-aminooctanoic acid. In one embodiment, the non-natural amino acid is N-acetylglucosamine-L-serine, N-acetylglucosamine-L- Threonine and O-phosphotyrosine. In one embodiment, the non-natural amino acid contains a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-serine -L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-aspartic acid, and O-mannosamine-L-serine. Examples of such amino acids also include those in which naturally occurring N- or O-bonds between amino acids and sugars are replaced by covalent bonds not commonly found in nature, including but not limited to alkenes, oximes, sulfur Ether, amide, etc. Examples of such amino acids also include sugars uncommon in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxygalactose, and the like. Specific examples of non-natural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-propargyloxyphenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthalene L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl -L-tyrosine (4-propyl-L-tyrosine), tri-O-acetyl-GlcNAc-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L -Phenylalanine, p-azido-L-phenylalanine, p-phenylalanine-L-phenylalanine, p-phenylalanyl-L-phenylalanine, L-phosphoserine, phosphoserine, phosphatide Amino acid, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine and isopropyl-L-phenylalanine, etc. Additional non-natural amino acids are disclosed in Liu et al., Annu Rev Biochem, 79:413-44, 2010; Wang et al., Angew Chem Int Ed, 44:34-66, 2005; and International Application No.: 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/US2006/0 47822 and PCT /US2006/044682, the entire content of each of the documents is incorporated into the present invention by reference. In one embodiment, the one or more unnatural amino acids may be p-acetylphenylalanine (pAF).

In certain embodiments of the invention, anti-CD3 antibodies having at least one unnatural amino acid include at least one post-translational modification. In one embodiment, at least one post-translational modification includes attaching a molecule containing a second reactive group to at least one molecule containing a first reactive group using chemical methods known to those of ordinary skill in the art to be suitable for the specific reactive group. groups of unnatural amino acids, the molecules Including but not limited to water-soluble polymers, derivatives of polyethylene glycol, drugs, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, bioactive agents, small molecules, or the above or any other desired compounds or Any combination of substances. For example, the first reactive group is an alkynyl moiety (including but not limited to the non-natural amino acid p-propargyloxyphenylalanine, where the propargyl group is sometimes also referred to as an acetylene moiety), while the second reactive group The group is the azide moiety, and [3+2] cycloaddition chemistry is utilized. In another example, the first reactive group is an azide moiety (including, but not limited to, the non-natural amino acid p-azido-L-phenylalanine), and the second reactive group is an alkynyl moiety. In certain embodiments of the modified anti-CD3 antibody polypeptides of the invention, at least one non-natural amino acid (including but not limited to non-natural amino acids containing a ketone functionality) is used, which includes at least one post-translational modification, wherein The at least one post-translational modification includes a sugar moiety. In certain embodiments, post-translational modifications are performed in vivo in eukaryotic or non-eukaryotic cells. In other embodiments, post-translational modifications are performed in vitro. In another embodiment, post-translational modifications are performed in vitro and in vivo.

In one example, non-natural amino acids can be modified to incorporate chemical groups. In one embodiment, non-natural amino acids can be modified to incorporate a ketone group. One or more non-natural amino acids may contain at least one oxime, carbonyl, dicarbonyl, hydroxylamine group, or combinations thereof. One or more non-natural amino acids may contain at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic olefin, acyclic alkyne, cyclooctyne, aryl/alkyl azide, norbornyl alkene, cyclopropene, trans-cyclooctene or tetrazine functional groups or combinations thereof.

In some embodiments of the present disclosure, non-natural amino acids are site-specifically incorporated into antibodies, antibody fragments, or variants. In one embodiment, non-natural amino acids are site-specifically incorporated into an anti-CD3 antibody, antibody fragment or variant. Methods of incorporating unnatural amino acids into molecules such as proteins, polypeptides or peptides are disclosed in U.S. Patent Numbers: 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 9,637,41 1(all of each of them The contents are incorporated herein by reference), as well as the Examples of the present invention. through this field Known methods incorporate one or more unnatural amino acids. For example, cell-based or cell-free systems can be used, and auxotrophic strains can also be used instead of engineered tRNAs and synthetases. In certain embodiments, orthogonal tRNA synthetases are used, such as, for example, 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. Incorporation of one or more non-natural amino acids into an antibody or antibody fragment or variant may involve modifying one or more amino acid residues in the antibody or antibody fragment or variant. Modifying one or more amino acid residues in the antibody or antibody fragment or variant may 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 may include changing codons encoding amino acids to nonsense codons. Incorporating one or more non-natural amino acids into an antibody or antibody fragment or variant may include modifying one or more amino acid residues in the antibody or antibody fragment or variant to be present in the antibody or antibody fragment or variant. Generates one or more amber codons. One or more non-natural amino acids can be incorporated into an antibody or antibody fragment or variant in response to an amber codon. One or more non-natural amino acids can be position-specifically incorporated into an antibody or antibody fragment or variant. Incorporation of one or more unnatural amino acids into an antibody or antibody fragment or variant may include one or more genetic codes for orthogonal chemical reactivity relative to the typical twenty amino acids. Unnatural amino acids to position-specifically modify biologically active molecules or targeting agents. Incorporation of one or more unnatural amino acids may include the use of a tRNA/aminoacyl-tRNA synthetase pair, in response to one or more amber nonsense codons ) position-specific incorporation of one or more unnatural amino acids into a defined position in a biologically active molecule or targeting agent. Other methods of incorporating unnatural amino acids include, but are not limited to, those disclosed 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. Selective reactions can be mediated by one or more enzymes. In a non-limiting example, the selective reaction of one or more cysteine with a formylglycine-generating enzyme (FGE) can produce one or more formylglycines, such as Rabuka et al., Nature Protocols 7:1052-1067, 2012. One or more non-natural amino acids can participate in a chemical reaction to form a linker. Chemical reactions that form linkers may include bioorthogonal reactions. Chemical reactions that form linkers can include click chemistry. See, for example, WO2006/050262, which is incorporated by reference in its entirety.

Bioactive molecules/reagents:

The present invention discloses anti-CD3 antibodies or antibody fragments or variants thereof, which comprise a biologically active molecule linked to the antibody or fragment or variant via a non-natural amino acid. Biologically active molecules include, but are not limited to, small molecules or agents, non-peptide compounds, drugs, second proteins or polypeptides or polypeptide analogs or derivatives, antibodies or antibody fragments or variants, second bioactive agents, targeting agents, or Any combination of the above or any other desired compounds or substances. In one embodiment, the bioactive agent is involved in recruiting cytotoxic T cells to cells, including but not limited to cancer cells or tumor cells. In one embodiment, the bioactive molecule is a small molecule, such as, but not limited to, folic acid or its derivatives or analogs or 2-[3-(1,3-dicarboxypropyl)ureido]glutaric acid (DUPA) or its derivatives or analogues. In one embodiment, the bioactive molecule is folic acid or a derivative or analog thereof. The bioactive molecule may be selected from cell targeting molecules, ligands, proteins, peptides, peptoids, DNA aptamers, peptide nucleic acids, vitamins, substrates or substrate analogs, cholecystokinin B receptors, gonadotropins Hormone-releasing hormone receptor, somatostatin receptor 2, avb3 integrin, gastrin-releasing peptide receptor, neuron Neurokinin 1 receptor, melanocortin 1 receptor, neurotensin receptor, neuropeptide Y receptor and C-type lectin 1 like molecule 1), receptor, co-receptor, transmembrane protein or cell marker or cell surface protein. Bioactive molecules can bind to target cells. Bioactive molecules can bind to cell surface proteins or cell surface markers or cell surface molecules on cells. Bioactive molecules can bind to cell surface molecules on cells including, but not limited to, cancer or tumor cells or immunosuppressive cells. The cell surface molecule may be a folate receptor molecule. The bioactive molecule may be an agent that binds prostate-specific membrane antigen (PSMA), such as, but not limited to, DUPA or analogs or derivatives thereof. Bioactive molecules or agents can bind to cells that overexpress or highly express cell surface markers, proteins, or receptors.

In one embodiment, the bioactive molecule may be folic acid or a folic acid ligand or an analog or derivative thereof. Bioactive molecules can bind to the folate receptor protein (FR). Such bioactive molecules may be N-(4-{[(2-amino-4-oxo-1,4-dihydropterin-6-yl)methyl]amino}benzoyl)- L-glutamic acid (folic acid), or its analogs or derivatives. Analogues thereof may be based on folic acid and retain the FR binding moiety. Folic acid analogues retain a significant portion of the folate structure. Furthermore, the folate analog can be a slightly modified form of folic acid because it is conjugated to a linker or an antibody or an antibody fragment or variant. For example, folate analogs may be slightly modified due to conjugation of folate carboxyl groups to linkers or antibodies or antibody fragments or variants. Additionally, due to the conjugation of folic acid to linkers or antibodies or antibody fragments, folic acid can be slightly modified but still maintain its FR binding properties. In one embodiment, the folate molecule targets folate receptor alpha. In one embodiment, the folate molecule targets folate receptor beta. In one embodiment, folic acid or folates are used as bioactive molecules or targeting agents that bind folate receptor (FR) antigens that are overexpressed or highly expressed on FR+ cell lines. In one embodiment, the cells are cancer cells or immunosuppressive cells, but are not limited thereto.

A bioactive molecule or agent can be site-specifically linked to one or more non-natural amino acids of an antibody or antibody fragment or variant via one or more linkers. The linker can be a chemical linker, a peptide linker, or a combination thereof. Chemical linkers include linkers via unnatural amino acids. Chemical connectors include Linker via one or more unnatural amino acids. Chemical linkers may include chemical conjugates. Peptide linkers include any amino acid sequence that joins two amino acid sequences. The peptide linker may comprise 1 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more Many, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. The peptide linker can be part of any antibody, including domains of the antibody, such as variable domains, CH1, CH2, CH3 and/or CL domains. Antibodies or antibody fragments or variants can be conjugated to biologically active molecules. Antibodies or antibody fragments or variants can be conjugated to biologically active molecules via chemical and/or peptide linkers. In one embodiment, the invention provides anti-CD3 antibodies or antibody fragments or variants conjugated to one or more biologically active molecules or agents. In one embodiment, one or more bioactive molecules or agents are one or more small molecules. In one embodiment, the invention provides anti-CD3 Fab antibodies or antibody fragments or variants conjugated to one or more small molecules. In one embodiment, the invention provides anti-CD3 Fab antibodies or antibody fragments or variants conjugated to one or more folate molecules. In one embodiment, the invention provides anti-CD3 Fab antibodies or antibody fragments or variants conjugated to one or more DUPA molecules. In one embodiment, one or more folate molecules and/or one or more DUPA molecules can be conjugated to the anti-CD Fab antibody via chemical and/or peptide linkers.

PEG linkers/ligands/conjugates:

Anti-CD3 antibodies or antigen-binding polypeptides and small molecules can be joined via linkers, polymers, or covalent bonds. The linker, polymer or small molecule itself can contain functional groups that are non-reactive towards 20 common amino acids. The linker or polymer may be a bifunctional linker or polymer. Bifunctional linkers or polymers are branched linkers or polymers. The bond or bonds involved in conjugating 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 unstable under desired conditions. The one or more bonds involved in joining the anti-CD3 antibody or antigen-binding polypeptide to the molecule via a linker, polymer, or covalent bond may allow for the regulated release of the antigen-binding polypeptide or other molecule. this field Technicians can produce a wide variety of small molecules through chemical means, isolation as natural products, or other means.

The present invention discloses 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 . An anti-CD3 antibody or antibody fragment or variant comprising a non-naturally encoded amino acid can be linked to two water-soluble polymers, such as two polyethylene glycol (PEG) molecules or ligands. Antibodies or antibody fragments or variants comprising non-naturally encoded amino acids and one or more biologically active molecules can be linked to one or more water-soluble polymers, such as polyethylene glycol (PEG) molecules or linkers. In one embodiment, an antibody or antibody fragment or variant comprising a non-naturally encoded amino acid and two biologically active molecules is linked to two water-soluble polymers, such as polyethylene glycol (PEG) molecules or linkers.

The method may include linking the antibody or antibody fragment to a bioactive molecule or a water-soluble polymer or a conjugate comprising a bioactive molecule and a water-soluble polymer. The method may include conjugating one or more linkers to a bioactive molecule to create a bioactive molecule-linker intermediate, and conjugating the intermediate to an antibody or antibody fragment. The method may include conjugating one or more linkers to a PEG molecule to produce a PEG-linker intermediate, and conjugating the PEG-linker intermediate to an antibody or antibody fragment. The method may include conjugating one or more linkers to an antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate, and co-conjugating the antibody-linker intermediate or antibody fragment-linker intermediate. Conjugated to another bioactive molecule, or a water-soluble polymer, or a conjugate comprising a bioactive molecule and a water-soluble polymer. Methods disclosed herein may include conjugating one or more linkers to one or more antibodies or antibody fragments, one or more biologically active molecules, or combinations thereof to produce one or more intermediates, such as antibody-linker intermediates , antibody fragment-linker intermediates and/or bioactive molecule antibody conjugates-linker intermediates. The method may include conjugating a first linker to an antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate. The method may include conjugating a linker to a bioactive molecule to produce a bioactive molecule-linker intermediate.

Methods of producing bispecific anti-CD3 antibody conjugates of the invention may comprise (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 with a biologically active molecule to produce a biologically active molecule-linker intermediate; and (c) linking two intermediates together to produce an anti-CD3 antibody-bioactive molecule conjugate; Biology Active molecules may be small molecules, including but not limited to folic acid or DUPA molecules or analogs or derivatives thereof. In certain embodiments, methods of producing bispecific anti-CD3 antibody conjugates of the invention may comprise: (a) combining a first linker with one or more non-natural amine groups comprising one or more non-natural amine groups incorporated into the antibody or antibody fragment; conjugating an acidic antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate; (b) conjugating a second linker to a biologically active molecule to produce a biologically active molecule-linker intermediate; and (c) ) connects two intermediates together to create an anti-CD3 antibody-bioactive molecule conjugate; the bioactive molecule can be a small molecule, including but not limited to folic acid or DUPA molecules or analogs or derivatives thereof. Methods of producing bispecific anti-CD3 Fab antibody-folate conjugates of the invention may comprise: (a) combining a first linker with an anti-anti-CD3 Fab antibody-folate conjugate comprising one or more unnatural amino acids incorporated into the antibody or antibody fragment; Conjugate a CD3 Fab antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate; (b) conjugate a second linker to a biologically active molecule to produce a biologically active molecule-linker intermediate; and (c) ) linking two intermediates together to produce an anti-CD3 Fab antibody-bioactive molecule conjugate; the bioactive molecule comprising one or more folic acid or DUPA molecules or analogs or derivatives; the linker comprising one or Multiple chemical and/or peptide linkers; the linkers comprise one or more PEG molecules. The one or more PEG molecules are linear or branched PEG molecules. One or more branched PEG molecules are bifunctional linkers. The average molecular weight of PEG molecules is 5kDa, 10kDa, 20kDa, 30kDa, 40kDa, 50kDa or greater. The average molecular weight of PEG molecules is 5K, 10K or 20K.

Conjugation of one or more linkers to the antibody or antibody fragment or bioactive molecule can occur simultaneously. Conjugation of one or more linkers to the antibody or antibody fragment or bioactive molecule can occur sequentially. Conjugation of one or more linkers to an antibody or antibody fragment or bioactive molecule can be performed in a one-step process For example enzymatic conjugation or chemical steps or processes or reactions are performed. The conjugation of one or more linkers to the antibody or antibody fragment or bioactive molecule can be performed in a two-step process, such as two enzymatic conjugations or two chemical steps or two processes or two reactions. Conjugation of one or more linkers to an antibody or antibody fragment or bioactive molecule can be performed in a two or more step process, such as two or more enzymatic conjugation or chemical steps or processes or reactions.

As is well known to those skilled in the art, conjugation of intermediates to antibodies or antibody fragments or bioactive molecules or water-soluble polymers may include oxime chemistry to form oxime bonds and/or click chemistry ( click chemistry). An antibody or antibody fragment may contain one or more unnatural amino acids. Linking the antibody or antibody fragment to the intermediate may include formation of an oxime between the non-natural amino acid and the linker intermediate. Conjugating the linker to the antibody or antibody fragment or biologically active molecule or water-soluble molecule may include ionic bonds, covalent bonds, non-covalent bonds or the like between the linker and the antibody or antibody fragment or biologically active molecule or water-soluble molecule. combination. Conjugation of linkers to antibodies or antibody fragments or bioactive molecules or water-soluble molecules is known in the art. See, eg, Roberts et al., Advanced Drug Delivery Reviews 54:459-476 (2002).

Conjugating one or more linkers to an antibody or antibody fragment and/or bioactive molecule may include forming one or more oximes between the linker and the antibody or antibody fragment or bioactive 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 the 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 bioactive molecule.

Conjugating one or more linkers to an antibody or antibody fragment may include position-specific conjugation of one or more linkers to an antibody or antibody fragment. Position-specific conjugation may involve linking one or more linkers to non-natural amino acids of the antibody or antibody fragment. Attachment of one or more linkers to non-natural amino acids of the antibody or antibody fragment may involve the formation of an oxime. Attaching one or more linkers to a non-natural amino acid of the antibody or antibody fragment may include the formation of a sulfide. As a non-limiting example, linking one or more linkers to a non-natural amino acid of the antibody or antibody fragment may include reacting the hydroxylamine of the one or more linkers with an aldehyde or ketone of the amino acid. The amino acid may be a non-natural amino acid. As a non-limiting example, linking one or more linkers to a non-natural amino acid of the antibody or antibody fragment may include reacting a bromo derivative of the one or more linkers with a thiol of the amino acid. The amino acid may be a non-natural amino acid.

One or more PEGs or linkers may contain a disulfide bond linking two cysteine residues using conjugation chemistry known to those skilled in the art. (See eg ThioBridge ^™ technology, Abzena). Two or more PEG molecules or linkers may contain maleimide bridges connecting two amino acid residues. The two amino acids may be at the C-terminus of the antibody or antibody fragment. One or more linkers may comprise a maleimide bridge connecting two cysteine residues. The two cysteine residues may be at the C-terminus of the antibody or antibody fragment. In one embodiment, one or more PEGs may be C-terminal PEG conjugated. In one example, the C-terminal PEG molecule may not involve covalent disulfide bonds or linkages between heavy and light chain antibodies or antibody fragments. In one embodiment, two or more C-terminal PEG molecules can be covalently linked or cross-linked between the heavy and light chains of an antibody or antibody fragment. Such PEG conjugates or linkers are described in the present invention.

In one embodiment the invention discloses a folate-PEG linker comprising one or more folate and one or more PEG molecules. The one or more PEG molecules may be 5 kDa, 10 kDa, 15 kDa, 20 kDa or larger. PEG molecules encompass both linear and branched polymers and have an average molecular weight between 0.1 kDa and 100 kDa. In one embodiment, the polyethylene glycol molecule or linker has a molecular weight of about 0.1 kDa to about 100 kDa. In one embodiment, the polyethylene glycol molecule or linker has 0.1 kDa to 50 kDa molecular weight. In one embodiment, the polyethylene glycol molecule or linker is a branched polymer or linker. In one embodiment, the molecular weight of each branch of the polyethylene glycol branched polymer or linker is from 1 kDa to 100 kDa, or from 1 kDa to 50 kDa. PEG molecules are well known in the art, see for example Shearwater Corporation's catalog "Polyethylene Glycols and Derivatives for Biomedical Applications" (2001).

In certain embodiments the invention discloses anti-CD3 Fab-folate PEGylated conjugates comprising one or more PEG molecules. In certain embodiments the invention discloses anti-CD3 Fab-folate PEGylated conjugates comprising one or more C-terminal PEG molecules. In one example, the C-terminal PEG molecule may not involve covalent disulfide bonds or attachments between heavy and light chain antibodies or antibody fragments. In one embodiment, the C-terminal PEG molecule can be attached separately to the heavy and light chains of the antibody or antibody fragment. In one embodiment, two or more C-terminal PEG molecules can be covalently attached or cross-linked between the heavy and light chains of an antibody or antibody fragment. In one embodiment, two or more PEG molecules can be covalently attached or crosslinked via a maleimide bridge connecting two cysteine residues.

Pegylation can be used to improve the pharmacokinetics of the composition and modulate cytotoxicity. PEGylation of proteins can increase their serum half-life by delaying renal clearance because the PEG moiety adds a considerable hydrodynamic radius to the protein. The covalent attachment of the hydrophilic polymer polyethylene glycol (abbreviated as PEG) is useful for many bioactive molecules (including proteins, peptides, especially hydrophobic molecules) to increase water solubility, bioavailability, extend serum half-life, and extend Methods to treat half-life, modulate immunogenicity, modulate biological activity, or extend circulation time. PEG has been widely used in pharmaceuticals, artificial implants, and other applications where biocompatibility, nontoxicity, and nonimmunogenicity are important. Preferably, pegylation does not alter or only minimally alters the activity of the bioactive molecule. Preferably, the increase in half-life is greater than any decrease in biological activity. Rader et al., Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5396-400, which is incorporated by reference, describe the development of new technologies for small synthetic molecules by reacting them with universal antibody molecules. Methods to provide effector function and extend serum half-life. The complex described is composed of mAb 38C2, a catalytic antibody that mimics native aldolase, and The reactive lysine residues target the reversible covalent bonds between the diketone derivatives of the Arg-Gly-Asp peptidomimetic integrin. In addition to increasing the half-life of the peptidomimetic, the complex was shown to selectively retarget antibodies to the surface of integrin αvβ3 and αvβ5 expressing cells.

In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a water-soluble polymer such as polyethylene glycol (PEG) via the side chain of the non-naturally encoded amino acid. In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to folic acid, wherein the linkage is via the side chain of the non-naturally encoded amino acid. In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a folic acid derivative via the side chain of the non-naturally encoded amino acid. In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a water-soluble polymer such as polyethylene glycol (PEG), wherein the linkage is via the side chain of the non-naturally encoded amino acid. In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is linked to a water-soluble polymer derivative such as a polyethylene glycol (PEG) derivative, wherein the linkage is via the side of the non-naturally encoded amino acid. Chain implementation. In one embodiment, a water-soluble polymer-folate linker is provided and an anti-CD3 antibody comprising at least one or more non-naturally encoded amino acids is passed through the side chain of the one or more non-naturally encoded amino acids. Attached to folic acid and/or water soluble polymer and/or linker.

In one embodiment, the folate moiety is derived from the following structures, including the structures shown in Figures 12A-12F:

In one embodiment, the water-soluble polymer is polyethylene glycol. In one embodiment, the term polyethylene glycol includes any form of polyethylene glycol, including linear polyethylene glycol, branched polyethylene glycol, bifunctional polyethylene glycol, multi-arm polyethylene glycol, derivatized polyethylene glycol, Ethylene glycol and forked polyethylene glycol.

The molecular weight of polyethylene glycol can range from 1 kDa to 100 kDa. The molecular weight of polyethylene glycol may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 kDa. The molecular weight of polyethylene glycol may range from about 1 kDa to about 25 kDa, or from about 5 kDa to 20 kDa. The molecular weight of polyethylene glycol may be about 5 kDa, or about 10 kDa, or about 20 kDa. The molecular weight of polyethylene glycol can be 5kDa or 10kDa or 20kDa. The molecular weight of polyethylene glycol may be 5 kDa.

In one embodiment, the bifunctional water-soluble polymer-folic acid linker has the following structure:

Where: A has the following structure:

B is a divalent group connecting A and C; C and E are each independently selected from -alkylene-, -alkylene-C(O)-, -(alkylene-O)n'-alkylene Base-,-(alkylene-O)n'-alkylene-C(O)-,-(alkylene-O)n'-(CH2)n'-NHC(O)-(CH2)n '-C(Me)2-S-S-(CH2)n'-NHC(O)-(alkylene-O)n'-alkylene-, -(alkylene-O)n'-alkylene -U-alkylene-C(O)- and -(alkylene-O)n'-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); and wherein Y is selected from hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidines, imines, diamines, azides, keto-amines, keto-alkynes, alkynes, cycloalkynes and alkenediones.

In one embodiment, B is a substituted divalent heterohydrocarbyl residue. In one embodiment, the substituents include one or more carboxyl, ketone and/or amide functional groups. In one embodiment, the heteroatoms are selected from N, O, and S.

In one embodiment, B has the following structure:

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

In one embodiment, D has the following structure:

In one embodiment, C and E are each -(alkylene-O)n'-alkylene-. In one embodiment, each alkylene group is -CH2CH2-.

In one embodiment, n' is 1 to 20, or 1 to 10, or 1 to 5.

In one embodiment, F has the following structure:

where n is 2 to 10,000. In one embodiment, n is chosen such that the molecular weight of polyethylene glycol (PEG) is 1 kDa to 100 kDa. For example, the molecular weight may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 kDa. For example, the molecular weight may be between about 1 kDa and about 25 kDa, or between about 5 kDa and 20 kDa. For example, the molecular weight may be about 5 kDa, or about 10 kDa, or about 20 kDa. For example, the molecular weight may be 5 kDa or 10 kDa or 20 kDa. For example, the molecular weight may be 5 kDa.

In one embodiment, the bifunctional water-soluble polymer-folic acid linker has the following structure:

In one embodiment, an anti-CD3 antibody comprising at least one non-naturally encoded amino acid is linked to a water-soluble polymeric bifunctional PEG-folate linker and thus has the following structure:

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

In one embodiment, Z has the following structure:

Where: J is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cyclo Alkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkane Lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene (substituted heteroarylene), alkarylene, substituted alkylene, aralkylene or substituted aralkylene; G is optional and when present is selected from Linkers for the following: 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) Alkyl)-, -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)kN(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N (R')S(O)kN(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, - C(R')=N-N=, -C(R')2-N=N- and -C(R')2-N(R')-N(R')-, where each R' is independently is H, alkyl or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; R1 is H, amino protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R2 is OH , ester protecting group, resin, at least one amino acid, polypeptide or polynucleotide; wherein R1 and/or R2 are anti-CD3 antibodies; and R3 and R4 are each independently H, halogen, lower alkyl or substituted lower Alkyl, or R3 and R4 or both R3 groups optionally form cycloalkyl or heterocycloalkyl.

In one embodiment, Z has the following structure:

其中每個R17獨立地選自H，烷基，被取代的烷基，烯基，被取代的烯基，炔基，被取代的炔基，烷氧基，被取代的烷氧基，烷基烷氧基，被取代的烷基烷氧基，聚環氧烷，被取代的聚環氧烷，芳基，被取代的芳基，雜芳基，被取代的雜芳基，烷芳基，被取代的烷芳基，芳烷基，被取代的芳烷基，-(亞烷 基或被取代的亞烷基)-ON(R")2，-(亞烷基或被取代的亞烷基)-C(O)SR"，-(亞烷基或被取代的亞烷基)-S-S-(芳基或被取代的芳基)，-C(O)R"，-C(O)2R"或-C(O)N(R")2，其中每個R"獨立地是氫、烷基、被取代的烷基、烯基、被取代的烯基、烷氧基、被取代的烷氧基、芳基、被取代的芳基、雜芳基、烷芳基、被取代的烷芳基、芳烷基或被取代的芳烷基；每個Z1是鍵、CR17R17、O、S、NR'、CR17R17-CR17R17、CR17R17-O、O-CR17R17、CR17R17-S、S-CR17R17、CR17R17-NR'或NR'-CR17R17；每個R'是H、烷基或被取代的烷基；每個Z2選自鍵、-C(O)-、-C(S)-、任選被取代的C1-C3亞烷基、任選被取代的C1-C3亞烯基和任選被取代的雜烷基；每個Z3獨立地選自鍵、任選被取代的C1-C4亞烷基、任選被取代的C1-C4亞烯基、任選被取代的雜烷基、-O-、-S-、-C(O)-、-C(S)-和-N(R')-；每個T3是鍵、C(R")(R")、O或S；其條件是當T3是O或S時，R"不能是鹵素；每個R"是H、鹵素、烷基、被取代的烷基、環烷基或被取代的環烷基；m和p是0、1、2或3，其條件是m或p中的至少一者不是0； M2是

、

、

、

、

、

、

、

或

，其中(a)表示與B基團鍵合並且(b)表示與雜環基團內相應位置的鍵合； M₃是

、

、

、

或

，其中(a)表示與B基團鍵合並且(b)表示與雜環基團內相應位置的鍵合；M₄是

、

、

、

或

，其中(a)表示與B基團鍵合並且(b)表示與雜環基團內相應位置的鍵合；每個R19獨立地選自C1-C6烷基、C1-C6烷氧基、酯、醚、硫醚、胺基烷基、鹵素、烷基酯、芳基酯、醯胺、芳基醯胺、烷基鹵化物、烷基胺、烷基磺酸、烷基硝基、硫酯、磺醯酯、鹵磺醯基、腈、烷基腈和硝基；q是0、1、2、3、4、5、6、7、8、9、10或11；並且每個R16獨立地選自氫、鹵素、烷基、NO2、CN和被取代的烷基。 where J, G, R1, R2, R3 and R4 are as defined above, and where D has the following structure:

wherein each R17 is independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkyl Alkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, Substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R")2, -(alkylene or substituted alkylene base)-C(O)SR",-(alkylene or substituted alkylene)-SS-(aryl or substituted aryl),-C(O)R",-C(O) 2R" or -C(O)N(R")2, where each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted Alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; each Z1 is a bond, CR17R17, O, S , NR', CR17R17-CR17R17, CR17R17-O, O-CR17R17, CR17R17-S, S-CR17R17, CR17R17-NR' or NR'-CR17R17; each R' is H, alkyl or substituted alkyl; Each Z2 is selected from the group consisting of 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 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; the condition is that when When T3 is O or S, R" cannot be 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 is

,

,

,

,

,

,

,

or

, where (a) represents a bond with the B group and (b) represents a bond with the corresponding position within the heterocyclic group; M ₃ is

,

,

,

or

, where (a) represents the bonding with the B group and (b) represents the bonding with the corresponding position within the heterocyclic group; M ₄ is

,

,

,

or

, where (a) represents a bond with the B group and (b) represents a bond with the corresponding position within the heterocyclic group; each R19 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, ester , ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, arylamide, 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; and each R16 is independent Ground is selected from hydrogen, halogen, alkyl, NO2, CN and substituted alkyl.

The present invention provides an efficient method for selectively modifying proteins with PEG derivatives, which method involves converting non-genetically encoded amino acids (including but not limited to those containing 20 naturally incorporated amino acids) in response to selector codons. Amino acids with functional groups or substituents not found in the protein (including but not limited to ketone, azide, or acetylene moieties) are selectively incorporated into the protein and those amino acids are subsequently modified with a suitable reactive PEG derivative. Once incorporated, the amino acid side chains can be modified using chemical methods known to those of ordinary skill in the art to suit the specific functional groups or substituents present in the naturally encoded amino acid. A wide variety of known chemical methods are suitable for use in the present invention to incorporate water-soluble polymers into proteins. Such a method Including, but not limited to, the Huisgen [3+2] cycloaddition reaction (see, e.g., Padwa, A. Comprehensive Organic Synthesis, Vol. 4, Trost, B.M., Pergamon, ed., Oxford, pp. 1069-1109, 1991; and Huisgen, R. .1,3-Dipolar Cycloaddition Chemistry, Padwa, A., ed., Wiley, New York, pp. 1-176, 1984), which include but are not limited to acetylene or azide derivatives respectively.

Because the Huisgen[3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution, it can modify proteins with extremely high selectivity. By adding a catalytic amount of Cu(I) salt to the reaction mixture, the reaction can be performed at room temperature under aqueous conditions with excellent regioselectivity (1,4>1,5). (See, eg, Tornoe et al., Org. Chem. 67:3057-3064, 2002; and 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 with suitable functional groups or substituents including, but not limited to, azido or acetylene derivatives. These molecules can be added to unnatural amino acids having an ethynyl group (including, but not limited to, p-propargyloxyphenylalanine) or to unnatural amino acids having an azide group (including, but not limited to, p-azido), respectively. -phenylalanine).

The five-membered ring produced by the Huisgen[3+2] cycloaddition reaction is usually irreversible in reducing environments and is stable in aqueous environments for long periods of time without hydrolysis. Therefore, the active PEG or PEG derivatives of the present invention can be used to modify the physical and chemical properties of a variety of substances under demanding aqueous conditions. More importantly, because the azide and acetylene moieties are specific for each other (e.g., will not react with any of the 20 common genetically encoded amino acids), they can be used at extremely high selectivities. Modify a protein in one or more specific positions.

The present invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives having one or more acetylene or azide moieties and related hydrophilic polymers. PEG polymer derivatives containing acetylene moieties are highly selective for conjugation with azide moieties that have been selectively introduced into proteins in response to selector codons. Similarly, PEG polymer derivatives containing azide moieties are highly selective for conjugation with acetylene moieties that have been selectively introduced into proteins in response to selector codons.

More specifically, azide moieties include, but are not limited to, alkyl azides, aryl azides, and derivatives of these azides. Derivatives of alkyl azides and aryl azides may include other substituents as long as acetylene-specific reactivity is maintained. The acetylene moiety includes alkyl and aryl acetylenes and their respective derivatives. Derivatives of alkylenes and aryl acetynes may include other substituents as long as azide-specific reactivity is maintained.

Therefore, anti-CD3 antibodies are intended to include any polypeptide that exhibits the ability to specifically bind a target molecule or antigen. Any known antibody or antibody fragment is an anti-CD3 antibody.

In one embodiment, compositions of anti-CD3 antibodies comprising a non-natural amino acid, such as p-(propargyloxy)-phenylalanine, are provided. Various compositions comprising p-(propargyloxy)-phenylalanine and including, but not limited to, proteins and/or cells are also provided. In one aspect, the composition comprising the non-natural amino acid p-(propargyloxy)-phenylalanine further comprises an orthogonal tRNA. Non-natural amino acids can be bonded (including, but not limited to, covalently bonded) to orthogonal tRNA, including, but not limited to, covalently bonded to orthogonal tRNA via amine-acyl bonds, covalently bonded to orthogonal tRNA 3'OH or 2'OH of the terminal ribose of tRNA, etc.

Measurement of anti-CD3 antibody activity and affinity of anti-CD3 antibodies to their antigen or binding partner :

Anti-CD3 antibody activity can be determined using standard in vitro or in vivo assays. For example, cells or cell lines that bind anti-CD3 antibodies (including, but not limited to, cells containing native anti-CD3 antibody antigens or binding partners or cells that produce recombinant anti-CD3 antibody antigens or binding partners) can be used to monitor anti-CD3 antibody binding. . For non-PEGylated or PEGylated antigen-binding polypeptides containing non-natural amino acids, the affinity of the anti-CD3 antibody for its antigen or binding partner can be determined by using techniques known in the art such as BIAcore ^™ biosensors. (Pharmacia) or Octet (ForteBio) to measure.

Regardless of which method is used to generate anti-CD3 antibodies, the anti-CD3 antibodies must be assayed for biological activity. If appropriate, tritiated thymidine assays can be performed to determine the extent of cell division. However, other biological assays can be used to determine the desired activity. Bioassays such as measuring the ability to inhibit the biological activity of an antigen (eg, enzymatic, proliferative, or metabolic activity) also provide an indication of anti-CD3 antibody activity. Other in vitro assays well known to those skilled in the art can be used to determine biological activity. In general, bioactivity tests should provide analysis of the desired results, such as increased or decreased bioactivity (compared to unchanged anti-CD3 antibody), different bioactivity (compared to unchanged anti-CD3 antibody), receptor Affinity analysis, conformational or structural changes, or serum half-life analysis, depending on the biological activity of the antigen. Those skilled in the art will recognize other assays that can be used to test the desired end result.

Measurement of Potency, Functional In Vivo Half-Life and Pharmacokinetic Parameters :

An important aspect of the present invention is the extended biological half-life obtained by constructing anti-CD3 antibodies with or without conjugation of the anti-CD3 antibody to a water-soluble polymer moiety. The rapid decrease in serum concentrations of anti-CD3 antibodies makes it important to evaluate the biological response to treatment with conjugated and non-conjugated anti-CD3 antibodies and their variants. Preferably, the conjugated and non-conjugated anti-CD3 antibodies and variants thereof of the invention also have an extended serum half-life after intravenous administration such that they can be measured, for example, by an ELISA method or by a primary screening assay. In vivo biological half-life measurements were performed as described herein.

The pharmacokinetic parameters of antigen-binding polypeptides containing non-naturally encoded amino acids can be assessed in normal Sprague-Dawley male rats. Pharmacokinetic data for anti-CD3 antibodies have been well studied in several species and can be directly compared to data obtained for anti-CD3 antibodies containing non-naturally encoded amino acids.

The specific activity of the anti-CD3 antibodies according to the invention can be determined by various assays known in the art. The biological activity of the anti-CD3 antibody mutant protein or fragment thereof obtained and purified according to the present invention can be tested by methods described or mentioned in the present invention or known to those skilled in the art.

Administration and pharmaceutical composition :

Polypeptides or proteins of the invention (including but not limited to anti-CD3 antibodies, synthetases, proteins containing one or more unnatural amino acids, etc.) are optionally used for therapeutic purposes, including but not limited to with a suitable pharmaceutical carrier combination. Such compositions include, for example, a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. Formulated to suit mode of administration. In general, methods of administering proteins are well known in the art and can be applied to administering the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptides of the invention are optionally tested in one or more appropriate in vitro and/or in vivo animal disease models to determine efficacy, tissue metabolism and estimate dosage according to methods well known in the art. In particular, the activity, stability or other suitable measures of the non-natural amino acids and natural amino acid homologues of the present invention (including but not limited to those modified to include one or more non-natural amino acids) can be initially measured by the activity, stability or other suitable measures. Comparison of anti-CD3 antibodies with natural amino acid anti-CD3 antibodies), i.e. in relevant assays, to determine dosage.

Administration is by any route commonly used to introduce the molecule into final contact with blood or tissue cells. The non-natural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods of administering such polypeptides to patients in the context of the present invention are available, and although more than one route may be used to administer a particular composition, a particular route may generally provide a more direct and effective effect than another route or reaction.

The pharmaceutically acceptable carrier depends in part on the particular composition being administered and the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the invention.

The polypeptide compositions may be administered by a variety of routes, including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal routes. Compositions containing modified or unmodified non-natural amino acid polypeptides can also be administered via liposomes. Such routes of administration and appropriate formulations are generally known to those skilled in the art.

Anti-CD3 antibodies containing non-natural amino acids, alone or in combination with other suitable components, may also be formulated in an aerosol formulation (i.e., they may be "nebulized") for administration via inhalation. Aerosol formulations can be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration (e.g., by intraarticular (within a joint), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and nonaqueous isotonic sterile injectable solutions, which may contain anti- Oxidizing agents, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickening agents, stabilizing agents agents and preservatives. Packaged formulations of anti-CD3 antibodies may be presented in unit-dose or multi-dose sealed containers such as ampules and vials.

Parenteral administration and intravenous administration are preferred methods of administration. In particular, routes of administration that have been used for natural amino acid homologue therapies (including but not limited to those commonly used for EPO, GH, anti-CD3 antibodies, G-CSF, GM-CSF, IFN, interleukins, antibodies and/or any other pharmaceutical delivery proteins) and currently used formulations provide preferred routes of administration and formulations for the polypeptides of the invention.

In the context of the present invention, a dose administered to a patient is sufficient to produce a beneficial therapeutic response in the patient over time, or may include, but is not limited to, inhibition of infection by a pathogen or other appropriate activity, depending on the application. The dosage is determined by the efficacy of the particular carrier or formulation, the activity, stability or serum half-life of the non-natural amino acid polypeptide used, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dosage will also depend on the presence, nature, and extent of any adverse side effects that may accompany administration of a particular carrier, formulation, etc., in a particular patient.

In determining the effective amount of a carrier or formulation to be administered for the treatment or prevention of disease (including but not limited to cancer, genetic disorders, diabetes, AIDS, etc.), the physician evaluates circulating plasma levels, formulation toxicity , disease progression and/or (if relevant) the development of antibodies against unnatural amino acid polypeptides.

Doses administered to, for example, a 70 kg patient will typically be in a range equivalent to those of currently used therapeutic proteins, adjusted for the altered activity or serum half-life of the relevant composition. The carriers of the present invention may supplement the treatment condition by any known conventional therapy, including antibody administration, vaccine administration, cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers Application of agents, etc.

For administration, the formulations of the invention are administered at a rate determined by the LD-50 or ED-50 of the formulation in question and/or the observation of any side effects of various concentrations of unnatural amino acids, including, but not limited to, at a rate suitable for the patient. The quality and overall health of the rate of application. Administration may be via single or divided doses.

If a patient receiving an infusion of the formulation develops fever, chills, or muscle aches, he/she will receive an appropriate dose of aspirin, ibuprofen, acetaminophen, or other pain/fever control medication . Patients who experience infusion reactions such as fever, myalgia, and chills are premedicated with aspirin, acetaminophen, or drugs including but not limited to diphenhydramine 30 minutes before subsequent infusions. Meperidine is used for more severe chills and muscle aches that do not respond quickly to fever reducers and antihistamines. Depending on the severity of the reaction, slow or interrupt cell infusion.

The human antigen-binding polypeptides of the invention can be administered directly to mammalian subjects. Administration is by any route commonly used to introduce anti-CD3 antibodies into a subject. Anti-CD3 antibody compositions according to embodiments of the invention include those suitable for oral, rectal, topical, inhaled (including but not limited to via aerosol), buccal (including but not limited to sublingual), vaginal, parenteral (including But not limited to subcutaneous, intramuscular, intracutaneous intraarticular, intrapleural, intraperitoneal, intracerebral, intraarterial or intravenous), topical (i.e. cutaneous and mucosal surfaces, including airway surfaces) and transdermal administration, however most appropriate in any given situation The approach will depend on the nature and severity of the condition being treated. Administration can be local or systemic. Formulations of the compounds may be presented in unit-dose or multi-dose sealed containers such as ampoules and vials. The anti-CD3 antibodies of the invention can be prepared as a mixture of unit dose injectable forms (including but not limited to solutions, suspensions or emulsions) and pharmaceutically acceptable carriers. Anti-CD3 antibodies of the invention may also be administered by continuous infusion (using, including but not limited to, minipumps such as osmotic pumps), single bolus injection, or sustained release depot formulations.

Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions (which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic), and aqueous and non-aqueous solutions. Aqueous sterile suspensions (which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives). Solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously mentioned.

The pharmaceutical compositions of the present invention may include pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier depends in part on the particular composition being administered and the particular method used to administer the composition. Accordingly, there is a wide variety of suitable pharmaceutical composition formulations (including optional pharmaceutically acceptable carriers, excipients or stabilizers) of the present invention (see, e.g., Remington's Pharmaceutical Sciences, Vol. 17th edition, 1985).

Suitable carriers include buffers containing phosphates, borates, HEPES, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin. Proteins, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartic acid, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including 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.

Anti-CD3 antibodies of the invention, including those linked to water-soluble polymers such as PEG, may also be administered by or as part of a sustained release system. Sustained release compositions include, but are not limited to, semipermeable polymeric matrices in the form of shaped articles, including but not limited to films or microcapsules. Sustained release matrices include those derived from 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. Patent No. 3,773,919 ; EP 58,481), polyglycolide (polymer of glycolic acid), polylactide-co-glycolide (copolymer of lactic acid and glycolic acid), polyanhydrides, L-glutamic acid and γ-ethyl- Copolymer of L-glutamic acid (U. Sidman et al., Biopolymers, 22, 547-556.1983), polyorthoesters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, Polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyethylene propylene, polyvinylpyrrolidone and silicone. Sustained release compositions also include liposome-encapsulated compounds. Liposomes containing said compounds can be prepared by methods known per se: DE 3,218,121; (Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77:4030-4034, 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324). All references and patents cited are hereby incorporated by reference.

Liposome-encapsulated anti-CD3 antibodies can be prepared, for example, by methods described in: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad.Sci.U.S.A., 77:4030-4034, 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; . The composition and size of liposomes are well known or can be readily determined empirically by those skilled in the art. Some examples of liposomes are described in: Park JW et al., Proc. Natl. Acad. Sci. USA 92:1327-1331, 1995; Lasic D and Papahadjopoulos D (ed.): 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 references and patents cited are hereby incorporated by reference.

In the context of the present invention, the dose administered to a patient should be sufficient to elicit a beneficial response in the subject over time. Typically, the total pharmaceutically effective amount of an anti-CD3 antibody of the invention administered parenterally per dose ranges from about 0.01 μg/kg to about 100 μg/kg, or from about 0.05 mg/kg to about 1 mg/kg of patient body weight per day, but This depends on treatment judgment. Frequency of dosing also depends on therapeutic judgment and may be more or less frequent than commercially available anti-CD3 antibody products approved for human use. Generally, the bispecific antigen-binding polypeptides of the invention may be administered by any of the above-described routes of administration.

Therapeutic uses of the anti-CD3 antibody bioconjugates of the invention :

The anti-CD3 antibody polypeptides of the invention can be used to treat a variety of disorders. The anti-CD3 antibody compositions disclosed herein can be used to modulate immune responses. Modulation of the immune response may include stimulating, activating, increasing, enhancing, or upregulating the immune response. Modulation of the immune response may include containing, suppressing, preventing, reducing, or downregulating the immune response. The disclosed anti-CD3 antibody-folate compositions may have advantages in penetrating solid tumors, during which natural folate ligands target, for example, folate receptor alpha on tumors, and folate receptor beta on immunosuppressive cells. In one embodiment, the tumor is a liquid or solid tumor.

The present invention discloses methods of treating a condition in a subject using an anti-CD3 conjugate or pharmaceutical composition of the invention. In some cancers, overexpression of specific cell surface receptors allows small molecules or drugs to selectively target cancer cells while minimizing effects on healthy cells. For example, 2-[3-(1,3-dicarboxypropyl)-ureido]glutaric acid (DUPA), which targets the prostate cancer-specific membrane antigen (PMSA), can be combined with T cells. Cell surface antigen (anti-CD3) combined with antibody conjugation to selectively recruit or target cytotoxic T cells to kill prostate cancer cells. N-(4-{[(2-Amino-4-oxo-1,4-dihydropterin-6-yl)methyl]amino}benzoyl)-L-glutamic acid ( Folate) can also be used as a bioactive molecule to bind to folate receptor (FR) antigens that are overexpressed on FR+ cancer cell lines.

The invention provides methods of treating cancer by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. The cancer may be ovarian cancer, including but not limited to epithelial, stromal and germ cell tumors. Ovarian cancer can include fallopian tube cancer or primary peritoneal cancer. Such cancers are characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. Cancer can be treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. In one embodiment, the invention provides a method of treating genetic diseases, AIDs or diabetes by administering to a patient a therapeutically effective amount of an anti-CD3 antibody of the invention. In one embodiment, the anti-CD3 antibody or therapeutic agent can be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody comprises position-specifically incorporated non-naturally encoded amine groups acid, the amino acid optionally conjugated to two folate and two PEGylated molecules. In another example, the anti-CD3 Fab antibody is incorporated into the anti-CD3 Fab antibody via a side chain of a non-natural amino acid and conjugated to two folate and two PEGylated molecules.

The present invention provides anti-CD3 antibodies for use in treating diseases or conditions in cells expressing high numbers of folate receptors. The anti-CD3 antibodies of the invention are useful in the treatment of cancer, including but not limited to ovarian cancer, including but not limited to epithelial, stromal and germ cell tumors. Ovarian cancer can include fallopian tube cancer or primary peritoneal cancer. Such cancers are characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. Cancer can be treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The anti-CD3 antibody of the present invention is used to treat genetic diseases, AIDS or diabetes, but is not limited thereto. The anti-CD3 antibodies of the invention can be used in the manufacture of medicaments for the treatment of diseases or conditions in cells expressing high numbers of folate receptors. The anti-CD3 antibodies of the invention can be used to manufacture medicaments for the treatment of cancers, including but not limited to ovarian cancer, including but not limited to epithelial, stromal and germ cell tumors. Ovarian cancer can include fallopian tube cancer or primary peritoneal cancer. Such cancers are characterized by high expression of folate receptor alpha (FOLR1), such as ovarian cancer. Cancer can be treated by recruiting cytotoxic T cells to folate receptor-positive (FR+) tumor cells. The anti-CD3 antibody of the present invention can be used to manufacture drugs for treating genetic diseases, AIDS or diabetes, but is not limited thereto.

In one embodiment, the condition to be treated is cancer. The cancer may be, 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) cancer, or with Any of these cancer related cancers or diseases or conditions. Carcinomas are cancers that begin in epithelial cells, the cells that cover the surface of the body, produce hormones and make up glands. As non-limiting examples, cancers include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovary cancer, brain cancer, vaginal cancer, vulva 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 area cancer, small bowel cancer, endocrine Systemic cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethra cancer, renal pelvis cancer, ureteral cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma tumors, brainstem gliomas, and spinal cord axis tumors. In some cases, the cancer is skin cancer, such as basal cell carcinoma, squamous cell carcinoma, melanoma, non-melanoma, or actinic (solar) keratosis. In one embodiment, the cancer is any cancer with high expression of folate receptor alpha or beta. In one embodiment, the condition to be treated is a disease or disorder. The disease or disorder may be a pathogenic infection. Pathogenic infections can be bacterial infections. Pathogenic infections can be viral infections. The disease or disorder may be an inflammatory disease. The disease or disorder may be an autoimmune disease. An autoimmune disease can be diabetes. The disease or condition may be cancer. In one embodiment, the disease or disorder is any disease or disorder with high expression of folate receptor alpha or beta. The disease or disorder may be a pathogenic infection. Bioactive molecules can interact with cell surface molecules on infected cells. Bioactive molecules can interact with molecules on bacteria, viruses or parasites. Pathogenic infections can be caused by one or more pathogens. In some cases, illness The primary organism is a bacterium, fungus, virus or protozoa. Exemplary pathogens include, but are not limited to: Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Fusidella Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella Legionella), Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia , Salmonella, 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 viruses (e.g., hepatitis A, B, and C), herpes simplex viruses (types 1 and 2), cytomegalovirus, herpesvirus, HIV, influenza virus, measles virus, mumps virus, papillomavirus, parainfluenza virus, poliovirus, respiratory syncytial virus, rubella virus, and Varicella-zoster virus. Examples of diseases or conditions caused by viruses include, but are not limited to, colds, flu, hepatitis, AIDS, chickenpox, rubella, mumps, measles, warts, and polio. The disease or disorder may be an autoimmune disease or an autoimmune related disease. An autoimmune disorder can be a dysfunction of the body's immune system, which causes the body to attack its own tissues. Examples of autoimmune diseases and autoimmune-related diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune Hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, Behcet's disease, celiac sprue, Crohn's disease, dermatomyositis, eosinophilic fascia erythema nodosum, giant cell arteritis (temporal arteritis), Goodpasture's syndrome, Graves' disease, Hashimoto's disease, idiopathic platelet ITP, IgA nephropathy, adolescents Arthritis of the Year, Diabetes, Juvenile Diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, 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, Swiss Reiter's syndrome, relapsing polychondritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, autoimmune diseases of sperm and testicles, stiff man syndrome ( stiff person syndrome), Takayasu's arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener's granulomatosis.

The disease or disorder may be an inflammatory disease. Examples of inflammatory diseases include, but are not limited to, alveolitis, amyloidosis, vasculitis, ankylosing spondylitis, avascular necrosis, Basedow's disease, Bell's palsy, bursitis, Carpal tunnel syndrome, celiac disease, cholangitis, chondromalacia patella, chronic active hepatitis, chronic fatigue syndrome, Cogan's syndrome, congenital hip dysplasia, costochondritis, Crohn's syndrome Crohn's Disease, cystic fibrosis, De Quervain's tendinitis, diabetes-related arthritis, diffuse idiopathic skeletal hypertrophy, discoid lupus, Ehlers-Danlos syndrome (Ehlers-Danlos syndrome), familial Mediterranean fever, fasciitis, fibrositis/fibromyalgia, frozen shoulder, ganglion cyst, giant cell arteritis, gout, Graves' Disease, HIV Associated rheumatic disease syndromes, hyperparathyroidism-associated arthritis, infectious arthritis, inflammatory bowel syndrome/irritable bowel syndrome, juvenile rheumatoid arthritis, Lyme disease , Marfan's Syndrome, Mikulicz's Disease, mixed connective tissue disease, multiple sclerosis, myofascial pain syndrome, osteoarthritis, osteomalacia, osteoporosis and corticosteroid-induced osteoporosis, Paget's Disease, gyrus rheumatism, Parkinson's Disease, Plummer's Disease, polymyalgia rheumatica , multiple myeloma inflammation, pseudogout, psoriatic arthritis, Raynaud's Phenomenon/Syndrome, Reiter's Syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, sciatica (lumbar radiculopathy) ), scleroderma, scurvy, sickle cell arthritis, Sjogren's syndrome, spinal stenosis, spondylolisthesis, Still's Disease, systemic lupus erythematosus, Takayasu's disease (pulseless) disease, tendonitis, tennis elbow/golfer's elbow, thyroid-related arthritis, trigger finger, ulcerative colitis, Wegener's granulomatosis and Whipple's Disease.

Pharmaceutical compositions comprising anti-CD3 antibodies may be formulated at a strength effective for administration by various means to human patients experiencing disorders that may be affected by anti-CD3 antibody agonists or antagonists, such as, but not limited to, alone or Antiproliferative, anti-inflammatory, or antiviral agents are used as part of a condition or disease. The average amount of anti-CD3 antibodies may vary, especially based on the advice and prescription of a qualified physician. The exact amount of anti-CD3 antibody is a matter of priority depending on factors such as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The present invention also provides for the administration of a therapeutically effective amount of another active agent, such as, but not limited to, an anti-cancer chemotherapeutic agent or an immunotherapeutic agent. Based on the therapy utilizing anti-CD3 antibodies, one skilled in the art can readily determine the amount to be administered.

Example:

The following examples are provided to illustrate, but not to limit, the claimed invention.

Example 1:

Criteria for selecting preferred locations for incorporation of non-naturally encoded amino acids into anti-CD3 antibodies: This example illustrates how a three-dimensional structure consisting of two anti-CD3 antibody molecules, or a secondary structure of an anti-CD3 antibody, can be utilized Preferably, the tertiary or quaternary structure of the antigen-binding polypeptide CD3 is selected to introduce non-naturally encoded amino acids.

The following criteria are used to evaluate each position of an anti-CD3 antibody used to introduce a non-naturally encoded amino acid: Residue (a) should not interfere with the binding of either anti-CD3 antibody based on the three-dimensional structure or secondary structure of the anti-CD3 antibody. , Structural analysis of tertiary or quaternary structure, b) should not be affected by alanine or homologue scanning mutagenesis, (c) should be surface exposed and exhibit minimal van der Waals or hydrogen bonding interactions with surrounding residues, (d) may be on one or more exposed faces of the anti-CD3 antibody, (e) may be at one or more positions of the anti-CD3 antibody juxtaposed with a second anti-CD3 antibody or other molecule or fragment thereof, (f) shall are missing or variable in anti-CD3 antibody variants, (g) result in conservative changes when replaced by non-naturally encoded amino acids, (h) can modulate the anti-CD3 antibody itself by altering the flexibility or stiffness of the complete structure as desired or a configuration comprising dimers or multimers of one or more anti-CD3 antibodies, (i) which may be present in highly flexible regions or structurally rigid regions, and (j) which may or may not be present in complementarity determining regions ( CDR). Additionally, further calculations were performed using the Cx program (Pintar et al., Bioinformatics, 18, p. 980) on anti-CD3 antibody molecules to assess the degree of protrusion of each protein atom. As a result, in one embodiment, the non-naturally encoded encoded amino acid is substituted at one or more positions of the anti-CD3 antibody.

Example 2:

Expression of anti-CD3 antibodies containing non-naturally encoded amino acids in E. coli: an introduced translation system containing orthogonal tRNA (O-tRNA) and orthogonal amine acyl-tRNA synthetase (O-RS) was used to express proteins containing Anti-CD3 antibodies for non-naturally encoded amino acids. O-RS preferentially chelates O-tRNA amino groups using non-naturally encoded amino acids. In turn, the translation system inserts the non-naturally encoded amino acid into the anti-CD3 antibody in response to the encoded selector codon.

Transformation of E. coli with plasmids containing modified anti-CD3 antibody genes and an orthogonal amine acyl tRNA synthetase/tRNA pair specific for the desired non-naturally encoded amino acid allows the transfer of non-naturally encoded amines Position-specific incorporation of amino acids into anti-CD3 antibodies. Transformed E. coli grown at 37°C in medium containing 0.01-100 mM of specific non-naturally encoded amino acids express modified anti-CD3 antibodies with high fidelity and efficiency. Anti-CD3 antibodies containing non-naturally encoded amino acids are produced by Escherichia coli host Chief cells are produced in the periplasm as soluble proteins. Methods for purifying anti-CD3 antibodies are well known in the art and have been demonstrated by SDS-PAGE, Western blot analysis, or electrospray-ionization ion trap mass spectrometry, among others.

Expression/Suppression: Suppression with the non-naturally encoded amino acid p-acetylphenylalanine (pAF): Suppression of amber mutations was achieved in E. coli using standard protocols known in the art. Briefly, for periplasmic inhibition of antibody fragments in Escherichia coli (Fab), expression vector constructs encoding orthogonal tRNA synthetases (e.g., orthogonal caseins from M. jannaschii) are used. plastids of aminoacyl-tRNA synthetase (MjTyrRS) were transformed into E. coli host cells. Overnight bacterial cultures were diluted 1:100 into shake flasks containing LB medium (Luria-Bertani) or Superbroth and grown at 37°C to an OD of approximately 0.8. Fab expression was induced by adding p-acetylphenylalanine (pAF) to a final concentration of 4mM while simultaneously achieving amber codon suppression. Cultures were incubated overnight at 25°C.

Suppression with non-naturally encoded amino acid derivatives: Suppression of amber mutations with non-naturally encoded amino acid derivatives, such as pAF (aa 9.2), is accomplished in a similar manner to the above, except that the p-amine group is used Acid-specific orthogonal synthetases (eg, tyramine-tRNA synthetase (MjTyrRS) from Methanococcus jannaschii). For example, inhibition is achieved by adding aa9.2 (4mM) upon induction.

Cells were collected by centrifugation and resuspended in periplasmic release buffer (50mM NaPO4, 20% sucrose, 1mM EDTA, pH 8.0) supplemented with 100ug/ml lysozyme and incubated on ice for 30 minutes. After centrifugation, the antibody fragments in the supernatant were immobilized on ProBind beads (Invitrogen; Carlsbad, CA) with the aid of their His tags, the beads were washed extensively with binding buffer and the bound fragments were eluted from the beads with 0.5 M imidazole. . Purified fragments were dialyzed against storage buffer (50mM HEPES, 150mM NaCl, 10% glycerol, 5% sucrose, pH 7.8). For small-scale analysis of Fab fragments expressed in the periplasm, 15 ml of E. coli culture was collected by centrifugation and resuspended in 1 ml of lysis buffer (B-PER, Pierce Biotechnology; Rockford) supplemented with 10 ug/ml DNase. ,IL). mix the mixture Incubate at 37°C for 30 minutes, dilute to 1X in protein loading buffer (Invitrogen; Carlsbad, CA) and analyze by SDS-PAGE.

Example 3:

Design and construction of humanized anti-CD3 gene in pFUSE vector - Mouse monoclonal anti-CD3 antibody SP34 (Harvard BIDMC) was humanized. Based on computer analogy analysis and design, in addition to the mouse CDR sequences in the selected human framework scaffold, 3 variable heavy chains (vH1.0, vH1.1 and vH1.2) containing various mouse framework backmutations were carried out and Synthesis of 3 variable light chain genes (vL1.0, vL1.1, and vL1.2) (Genewiz, South Plainfield, NJ). Table 2 lists the mouse framework residues (backmutations) and their Kabat numbers that are retained in the 3 variable heavy chains (vH) and 3 variable heavy chains (vH) of the 9 humanized anti-CD3 Fabs mentioned in Figure 1 In the light chain (vL). Amino acid residues restored to the human framework sequence are shown in bold.

As shown in Table 2, there are 5 mouse backmutations in the variable light chain (V36, G46, G49, G57, and V58) and 4 mouse backmutations in the variable heavy chain (N30, A49, I77, and V93 ). The six short synthetic variable genes shown in Table 2 were cloned into Invivogen's heavy chain (HC) and light chain (LC) expression vectors (pFUSE-CHIg-HG1 and pFUSE-CLig-, respectively). hk), generating plasmids expressing humanized anti-CD3 SP34 antibodies. As shown in Figure 1 and Table 2, variable light chain vL1.0, in which all five mouse backmutations were converted to human residues, lost binding to any variable heavy chain (vH) combination.

Example 4:

Expression of humanized anti-CD3 antibodies in the HEK293 transient system: Human human peptides described in the previous examples were The SP34 antibody was transiently expressed into HEK293 cells. The protein concentration in the cell culture medium from each co-transfection experiment was determined and used directly in the PBMC-based CD3 binding assay without further purification. The controls used were a chimeric SP34 construct (positive control) and an irrelevant PSMA antibody construct (negative control). Humanized anti-CD3 antibodies without incorporation of non-natural amino acids and with or without HC-DKTHT extension were also expressed and characterized in HEK293 cells (Table 3). These antibodies are used to screen for low affinity Fab variants. Table 3 shows the novel wild-type (WT) amino acid sequences of humanized anti-CD3 heavy chain (vH+CH1) and light chain (vL+CL) sequences used in different combinations to generate the Fabs disclosed in the present invention WT sequence. Table 3 also shows the WT amino acid sequence of the humanized anti-CD3 heavy chain (vH+CH1-DKTHT) sequence.

Table 3 - Wild type (WT) amino acid sequence - anti-CD3 heavy chain (vH+CH1), (vH+CH1-DKTHT) and light chain (vL+CL)

Example 5:

Testing CD3 binding to activated human and cynomolgus monkey PBMC: To differentiate between the generated humanized anti-CD3 antibodies, activated human PBMC (n=2) were used to test binding to human CD3. Activation of PBMC and subsequent fluorescence-based CD3 binding assay followed as previously described (see, eg, Angew Chem Int Ed Engl, 52(46):12101-12104, 2013). As shown in Figure 1, three antibodies with vL1.0 light chains lost binding to human CD3 with any vH heavy chain combination. The remaining six humanized antibodies were titrated for binding to human and cynomolgus CD3 using activated human and cynomolgus PBMC, respectively. As shown in Figures 2A-2F, all 6 humanized anti-CD3 antibodies (engineered from the combination of 3 vH heavy chains and 2 vL light chains shown in Table 3) retained binding to both human and cynomolgus CD3 Quite a combination. Obtained as follows Six humanized anti-CD3 antibodies were obtained: through 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)) combinations produce Fab1, Fab2 and Fab3 wild type. Through LC ((vL1.1+CL); (SEQ.ID.NO: 8)) and HC ((vH1.2+CH1); (SEQ.ID.NO: 1)), ((vH1.1+CH1 The combination of ); (SEQ.ID.NO:2)) and ((vH1.0+CH1); (SEQ.ID.NO:3)) resulted in Fab4, Fab5 and Fab6 wild-type, respectively.

Since the data showed no significant differences between the 6 humanized antibodies with respect to binding to the two CD3 substrates, Fab1 (vH1.2+vL1.2 combination) was selected to further demonstrate key aspects of the invention. For example, Fab1 was used for expression in E. coli for further optimization using proprietary unnatural amino acid (UAA) incorporation technology via a previously described orthogonal amber suppression system (see e.g. WO2017/079272, WO2012/166560 and WO2013/192360). Table 4 shows a list of mouse framework backmutations necessary to retain binding activity in these 6 Fabs, Table 4 provides the mouse frameworks retained in the 6 humanized anti-CD3 Fabs mentioned in Figures 2A-2F List of residues (reverse mutations) and their Kabat numbers. Amino acid residues restored to the human framework sequence are shown in bold. The vL1.2 and vH1.2 variable chain combination (underlined) yielding Fab1 was selected as a precursor for further modifications.

Example 6:

Cloning of synthetic Fab genes into E. coli expression vectors: Synthetic Fab genes were cloned into proprietary standard E. coli expression vectors using the Gibson Assembly cloning kit (New England Biolabs). After sequence verification of each expression plasmid, each plasmid was transformed into the standard E. coli production host strain W3110B60 and isolated single colonies of each plasmid were purified into glycerol vials. The glycerol vials were used as production clones for E. coli fermentations of these Fab molecules. The amino acid sequences used to engineer the 4 heavy chains and 3 light chains of these Fabs are shown in Table 5 as SEQ. ID. NO: 10 to 20.

Three light chain (LL157pAF, LK172pAF and LS205pAF) pAF variants (SEQ.ID.NO: 18 to 20) and 3 double pAF variants ((HK129pAF+LL157pAF); (SEQ.ID.NO: 16 and 18)) ;((HK129pAF+LK172pAF);(SEQ.ID.NO:16 and 19)) and ((HK129pAF+LS205pAF)(SEQ.ID.NO:16 and 20)) were engineered. SEQ.ID.NO: 10 to 13 represent heavy chain pAF variants without the 5-aa heavy chain C-terminal extension (DKTHT).

E. coli fermentation : The fermentation process for the production of anti-CD3 Fab-pAF consists of two stages: (i) inoculum preparation and (ii) fermentor production. Inoculate from a single glycerol vial, thaw, dilute 1:1000 (v/v) into 50 mL of defined seed medium in a 250 mL baffled Erlenmeyer flask, and culture at 37°C and 250 rpm. The fermentor was cleaned and autoclaved before use. The specified amount of basal medium is added to the fermenter and steam sterilized. Before inoculation, add the specified amount of kanamycin sulfate solution, feed medium and P2000 antifoam agent to the basal medium. All solutions added to the fermentor after autoclaving are 0.2 μm filtered or autoclaved and then added aseptically.

The production fermenter was inoculated at a target OD600 of 0.0004 by aseptically transferring the contents of the inoculum. After inoculation, cultures were sampled at appropriate intervals to determine OD600. Temperature, pH and dissolved oxygen are monitored and controlled at designated set points of 37°C, 7.0 and >30% respectively. The pH is controlled by adding ammonium hydroxide solution or sulfuric acid. Dissolved oxygen is controlled by varying the stirring speed and by increasing the oxygen composition in the injected air/oxygen mixture. Add antifoaming agents during fermentation to control foaming.

When the cell density reaches OD600>25, add a dollop of feed medium. When the cell density reached OD600>50, feed medium was added at a constant flow rate of 0.094 mL/L starting volume/min for 32 h until it was reduced to 0.052 mL/L starting volume/min at the end of fermentation. Immediately after feed initiation, a specified amount of a solution of a non-naturally encoded amino acid (eg, pAF) is added aseptically to incorporate the non-natural amino acid into the protein amino acid sequence. At the same time, the temperature was changed from the 37°C used during growth to 27°C for production. Production is controlled by the phoA activator and begins when phosphate levels in the culture medium are depleted. Collection began approximately 48 hours after induction.

Example 7:

Purification and conjugation of folate and PEG-folate: The anti-CD3 Fab of the invention was produced in E. coli cells and recovered from whole cell lysate (WCL) supernatant. Cell lysis was performed at 4°C. Cells were lysed in 100mM acetic acid, 100mM NaCl, 1mM EDTA (pH 3.5) in a volume equal to the original fermentation volume, resulting in a post-lysis pH of 4.1-4.2.

After lysis, WCLs were centrifuged at 15,900 x g for 30 min at 4°C and filtered (0.8/0.2 μm) to remove precipitated proteins and cell debris. Anti-CD3 Fab was 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) was used as a polishing column to separate the 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 was then buffer exchanged to 50mM acetate, 5% trehalose, pH 4.0 and concentrated in preparation for incubation with folic acid or PEG-folic acid conjugation. This step was performed at 4°C using an Amicon Ultracel 10K regenerated cellulose (15 mL) centrifuge device.

After buffer exchange and concentration to 50mM acetate, 5% trehalose, pH 4.0, anti-CD3 Fab is conjugated to folate or PEG-folate to target folate receptors on cancer cells. The conjugation reaction was carried out at 28°C and pH 4 for 24-48 hours. After conjugation with folate, anti-CD3 Fab-folate buffer was exchanged into formulation buffer, 50mM histidine, 100mM NaCl, 5% trehalose, pH 6.0. This step was performed at 4°C using an Amicon Ultracel 10K regenerated cellulose (15 mL) centrifuge device. After conjugation with PEG-folate, unconjugated, monoconjugated, and doubly conjugated anti-CD3 Fab-PEG-folate were separated using Toyo SP 5PW cation exchange chromatography.

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 produced. Add the desired folate-PEG (5K, 10K, 20K, 5K2 or 10K2 PEG) to the buffered solution of CD3 Fab (50mM acetate, 5% trehalose, pH 4) at 28°C. After one hour, the mixture was purified by cation exchange chromatography (Toyo SP 5PW) and formulated with 50mM histidine, 100mM NaCl, 5% trehalose, pH 6.0 buffer by using a centrifugal screening program (c/o 10K) , to obtain the desired CD3 Fab-folate-PEGylated composition. The structure, chemistry and conjugation of CD3 Fab folate conjugates are described herein and shown in Figures 12A-12D.

Additionally, a CD3 Fab folate-PEGylated C-terminal conjugate was generated. To CD3 folate (8.0 mg) in PBS (2.0 mL), EDTA (6.7 uL, 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 through a desalting column using 5 mmol EDTA in PBS. 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)2 C-terminal conjugate, CD3 Fab folate-(PEG10K)2 C-terminal conjugate and CD3 Fab folate-(PEG20K)2 C-terminal conjugate. The structure, chemistry and conjugation of C-terminal PEG conjugates are described herein and shown in Figures 12E-12F.

Example 8:

In vitro binding and killing assays: Purified humanized anti-CD3 Fabs conjugated with folic acid at different heavy chain positions (HA114, HS115, HK129, HT160) were initially tested for binding to both human and cynomolgus CD3 in these The corresponding unconjugated protein with pAF in position as well as the wild-type protein serves as a control. Table 6 depicts the EC50 of modified anti-CD3 Fab1 protein purified from E. coli cells for activation of human and cynomolgus monkey PBMC. As shown in Table 6, neither pAF nor conjugated folate at different positions significantly interfered with its CD3 binding.

The cytotoxicity of these folate-conjugated anti-CD3 Fabs was tested using activated human and cynomolgus monkey PBMC with SKOV-3 cells at E:T=10:1 with and without 50 nM serum folate (SFA). Cytotoxicity assays were performed as previously described (see, eg, Angew Chem Int Ed Engl, 52(46):12101-12104, 2013). Briefly, various E:T ratios of effector cells (activated PBMC or non-activated PBMC) and target cells (SKOV-3, KB, etc.) were mixed with different concentrations of anti-CD3 Fab in U-shaped bottom 96-well plates. -Folic acid co-culture overnight or as indicated. The amount of LDH released into the culture medium was used as Indicators of cytotoxicity and were measured using a non-radioactive cytotoxicity assay kit from Promega following the manufacturer's instructions.

As shown in Table 7, there were no significant differences in killing EC50 between folate conjugation positions. However, when 50 nM SFA was present, an approximately 15-60-fold decrease in cytotoxicity EC50 was observed compared to no SFA due to the competitive inhibitory effect of free SFA. Furthermore, for each variant, the cynomolgus monkey EC50 was approximately 10 times higher than the human EC50.

Example 9:

Cytotoxicity of anti-CD3-folate variants against multiple FOLRα tumor cell lines with different levels of folate receptor (FRα): in vitro cytotoxicity assay using 6 different cell lines with different levels of FRα overexpression on the cell surface , three different anti-CD3 Fab-folate conjugates were tested for activity at positions HA114, HS115 and HK129 (Table 8). The number of FRα ranges from 5,700 per cell in the alveolar basal epithelial cell carcinoma A549 cell line to 1,630,000 per cell in the nasopharyngeal carcinoma cell line. cells with activated human peripheral blood mononuclear cells (PBMC; target:effector cell ratio 1:10) Co-cultured and treated with various concentrations of anti-CD3 Fab-folate in the presence of 50 nM SFA. Cytotoxicity was quantified by measuring the levels of LDH released from lysed cells and using CellTiter-Glo and FACS-based toxicity assays.

Anti-CD3 Fab-folate conjugates at all three different conjugation positions were shown to effectively kill all 5 cell lines with FRα above 10K. However, no killing effect of the conjugates at any position was observed on the A549 cell line with FRα of 5,700. The effective killing threshold appears to be approximately 10,000 FRα; however, above this 10K threshold, killing activity appears to be independent of FRα number. Since the in vitro killing EC50 did not change significantly at the folate conjugation position in the antibody, the HK129 position was selected as the folate conjugation position for further experiments.

Example 10

This example shows the effect of folate-PEG conjugation at a single position or the addition of a second folate (bifolate) on the in vitro binding affinity and cytotoxic activity of various anti-CD3 Fab1-HK129-pAF molecules.

Effect of folic acid-PEG conjugation: Table 9 shows the effect on binding after conjugating 5K or 20K linear PEG molecules with folic acid using bifunctional linkers as described in the present invention and Table 10 shows cytotoxicity. Depending on the size of 5K or 20K PEG, a modest reduction in binding (1.7- to 8.4-fold) was observed for both activated human and cynomolgus monkey PBMC. However, for both human and cynomolgus monkey PBMC, cytotoxic activity was greatly reduced with 20K PEG compared to 5K PEG (3.7 to 5.6-fold vs. 50 to 58-fold). Based on this experiment, 5K PEG was used instead of 20K PEG to extend half-life.

Effect of double folate (bifolate) conjugation: Table 11 shows the effect of a second folate conjugate at two light chain positions (LL157 and LK172) combined with heavy chain position HK129 on in vitro binding, And Table 12 shows the cytotoxicity. Sure enough, a modest increase in both binding affinity and cytotoxic activity was observed for the bifolate variant compared to the HK129 monofolate molecule. A modest increase in the cytotoxicity of bifolate molecules relative to monofolate molecules was also evident in the presence or absence of 50 nM SFA. Based on this experiment, the LL157 position is superior to the LK172 position for combination with the HK129 position for future research.

Summary of binding affinity and in vitro cytotoxicity: Table 13 summarizes the effects on the binding and cytotoxic activity of various anti-CD3 Fabs due to PEGylation and secondary folate conjugation. A modest decrease in binding affinity to both CD3 and FRa results in a significant decrease in potency and is related to the size of the PEG. Despite the decrease in CD3 binding affinity, conjugation of bifolate molecules increases the affinity for FRα, thereby increasing the cytotoxic potency. Despite the reduced affinity for both targets due to PEG conjugation, the in vitro killing EC50 of all CD3-Fab-folate molecules remained high in the range of 37 to 335 pM.

Example 11

Effect of various effector to Target (E:T) cell ratios on the cytotoxicity of anti-CD3 Fab1 molecules on SKOV-3 cells: To test the effects of E:T ratios on SKOV-3 Cytotoxicity assay of 3 anti-CD3 Fab1 molecules was performed in the presence of 50 nM SFA at E:T ratios of 10:1, 5:1, 1:1, 1:5 and 1:10. As expected and shown in Table 14, the E:T ratio correlated with in vitro killing potency. Under E:T=1:1, observe A 2-fold increase in the EC50 of the difolate molecule was measured, while an 8-fold decrease in the EC50 of the 5KPEG-folate molecule was detected. All three molecules remained highly potent within E:T ratios of 10:1 to 1:10, with EC50s ranging from 1.69 to 175pM.

Example 12:

Pegylation of anti-CD3 Fab1 molecules to extend plasma half-life: The pharmacokinetic properties of anti-CD3 Fab1-folate conjugates in rats were studied in the presence and absence of PEG conjugation. Male Sprague Dawley rats (approximately 7 weeks old) were used. On the day of dosing, the body weight of each animal was measured. Samples of 1 mg of each unconjugated and conjugated anti-CD3 antibody per kg body weight were injected intravenously into the tail vein of three rats. At different time points after injection, 500 μl of blood was drawn from each rat under CO2 anesthesia. Blood samples were stored at room temperature for 1.5 hours, followed by serum separation by centrifugation (4°C, 18000 x g, 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 antibodies in the serum samples was quantified by an in vitro activity assay of anti-CD3 antibodies.

As shown in Figure 3, serum concentrations of both anti-CD3 Fab1-folate and the corresponding unconjugated anti-CD3 Fab1 decreased rapidly at the same rate, with serum half-lives of less than one hour. In contrast, the serum half-life of both PEG-conjugated anti-CD3 Fab1 was significantly extended to 6.1 hours and 10.6 hours for 5KPEG and bis-5KPEG, respectively. Table 15 shows IV administration of various anti-CD3 Fab1 molecules in rats Various PK parameters after. This data shows that bis-5KPEG-bifolate prolongs T1/2 and significantly increases AUC (13.2-fold increase over Fab1-folate and 3.9-fold increase over Fab1-5KPEG-folate) (Table 15).

Example 13

This example shows the generation and screening of anti-CD3 low affinity antibody variants in HEK293 cells.

De-convolution of mouse backmutation and germline mutations: mouse framework residues (backmutation) of both vH and vL sequences obtained during humanization of anti-CD3 antibodies, passed one at a time were restored to human germline residues to examine their effect on antigen binding (deconvolution). for vH sequence, four mouse framework residues (backmutations) at Kabat positions 30, 49, 77, and 93 are predicted to play a key role in antigen binding (Tables 2-3 and 16-17). Similarly, for the vL sequence, five mouse framework residues (backmutations) at Kabat positions 36, 46, 49, 57, and 58 are also predicted to play a critical role in antigen binding.

To evaluate the impact on antigen binding (deconvolution), 4 new vL (vL2.1 to vL2.4) and 3 new vH (vH2.1 to vH2.3) plastomes were generated (Table 16- 1st round plastid in 17). These vH and vL plasmids were used in HEK293 cell co-transfection experiments together with the original vH and vL plasmids described in the above examples (round 0 plasmids in Tables 16-17). A total of 35 transient transfections were performed in Round 1 of screening, and cell culture supernatants were used directly to screen for binding as described below and in the Examples above.

Based on the results of Round 1 screening, 3 additional vH plasmids (vH2.4 to 2.6) were generated (Round 2 plasmids in Tables 16-17) to evaluate the additive effect of individual back mutations. In a similar manner, a new vL plasmid (vL2.5) was generated by combining two mouse backmutations. Additionally, mouse germline mutations present in the vH and vL CDRs conferring reduced binding in this round were analyzed. To this end, 3 new vH plastoms (vH3.1 to 3.3) were generated by changing the amino acids at Kabat position N35 in HC-CDR1 and Y52c in HC-CDR2 (the second round plasmid in Table 16-17 body). In a similar manner, the vL plasmid (vL3.1) (round 2 plasmid in Tables 16-17) was generated by changing the amino acid at Kabat position K53 in LC-CDR2. A second round of screening experiments for a total of 43 transfections was performed by selectively combining various vL/vH plasmid pairs.

Screening for human and cynomolgus CD3 binding: During the screening phase, HEK293 culture supernatants were used directly to test binding to human CD3. In the first round of experiments, 35 clones were initially tested for binding to human CD3 at three different protein concentrations, and 13 clones were selected for detailed binding assays to both human and cynomolgus CD3. Eleven-point binding titration curves were generated for each clone for both human (Fig. 4A) and cynomolgus CD3 (Fig. 4B). Based on the data, Fabs 7 to 10 were selected as low-affinity candidates for further evaluation (Figures 4A-4B). Similarly, 43 clones were screened in the second round of experiments. exist After performing the two-stage screening as described above for the first round, 11 low-affinity variants were obtained, 4 of which are shown in Figures 5A-5B.

These 15 low affinity anti-CD3 Fab variants (Fab 7-10 from round 1 and Fab 11-21 from round 2) were then transferred into an E. coli expression system for non-natural amino acids ( UAA) and further tested as described in the examples below. Figure 6A shows a Fab-folate conjugate of the same wild-type Fab as shown in Figure 4A. Fab-folate conjugates representing wild-type Fab obtained from round 2 screening are depicted in Figure 6B. Table 18 shows a list of low affinity mutations along with their HC, LC and Fab IDs. As shown, both mouse framework revertants and germline CDR mutations play important roles in conferring reduced binding to both human and cynomolgus monkey CD3. In the variable light chain (vL), five mouse reversion residues at Kabat positions V36, G46, G49, G57, and V58 and one mouse germline residue at Kabat position K53 in LC-CDR2 were implicated in reduced binding . In the variable heavy chain (vH), four mouse reversion residues at Kabat positions N30, A49, I77 and V93 in HC-CDR1 and two mouse residues at Kabat position N35 and position Y52c in HC-CDR2 Germline residues implicated in reduced binding.

SEQ.ID.NO: 21 to 29 represent the amino acid sequences of the heavy chain with a 5-aa C-terminal extension (DKTHT) used in low affinity variant Fab screening. SEQ.ID.NO: 30 and 38 represent these humanized heavy chain amino acid sequences without the 5-aa heavy chain C-terminal extension. SEQ.ID.NO:39 represents the amino acid sequence of the light chain used in screening low-affinity variant Fab in HEK293 cells.

Example 14:

This example shows the construction, expression, purification and testing of low affinity humanized anti-CD3 Fab variants generated from E. coli cells using the heavy chain HK129 amber mutation.

Cloning into E. coli expression vectors: Synthetic genes were designed for all low affinity variants disclosed in Table 20, SEQ.ID NO:40-62 with STII-LC-spacer-STII-HC expression cassette structure, in the heavy chain The amber TAG stop codon was inserted at position HK129 and cloned into a proprietary E. coli expression vector as described in the examples above. The amino acid sequences of the heavy and light chains used to engineer these Fabs are shown as SEQ. ID. NO: 40 to 62. SEQ.ID.NO: 40 to 48 represent the humanized heavy chain HK129 pAF variants with a 5-aa heavy chain C-terminal extension (DKTHT) used in the tests. SEQ.ID.NO: 49 and 57 represent these humanized heavy chain HK129 pAFs without the 5-aa heavy chain C-terminal extension (DKTHT) Variants. SEQ.ID.NO:58-62 represent light chain sequences used in combination with the HK129pAF-DKTHT variant of SEQ.ID.NO:40 to 48, which were expressed in E. coli and further characterized.

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

Binding of Folate-conjugated Fabs to Human and Cynomolgus CD3: Binding of low-affinity variants of E. coli-produced and folate-conjugated humanized anti-CD3 Fabs was performed as described in the Examples above. Figures 6A-6B show the binding affinities for human CD3 of two subsets of low affinity Fab variants of anti-CD3 Fab-HK129-Folate and the positive control Fabl. Of the low-affinity Fabs tested, 10 failed to bind significantly to human CD3, even at the highest concentration tested (1000 nM). The remaining four Fabs (Fabs 7 to 10; described in Table 18) had binding EC50s ranging from 23.4 nM to 83.6 nM compared to 2.31 nM for control Fab1. Fab 21 shows weak binding activity and does not saturate even at a concentration of 1000 nM. As shown in Figures 7A-7B, very similar binding profiles were observed for cynomolgus monkey CD3 binding.

Cytotoxicity assay of folate-conjugated Fab using human and cynomolgus monkey PBMC: Cytotoxicity of low-affinity Fab was performed as described in the examples above. Figure 8 shows cytotoxicity data of activated human PBMC using SKOV-3 cells. As shown, all 4 Fabs with 10- to 36-fold reduced binding affinity to human CD3 (Figures 6A-6B) showed comparable killing activity compared to the control Fabl (Figure 8). Three Fabs (Fab 11, 19 and 20) did not show any cytotoxic activity (data not shown). All other 8 Fabs showed considerable killing activity, but they failed to bind even at 1000 nM concentration. Very similar cytotoxicity profiles were observed using activated cynomolgus monkey PBMC (Figure 9).

Example 15:

This example shows T cell activation and cytokine release assays of humanized anti-CD3 low affinity antibody variants.

T cell activation/cytokine release assay: Purified human T cells and T cell depleted from the same volume of whole blood by EasySep Human T Cell Enrichment Kit and EasySep Human CD3 Positive Selection Kit (STEMCELL Technologies Inc), respectively. of human PBMC. Confirm the purity of isolated T cells and helper cells by flow cytometry. To selectively monitor T cell activation in the presence of helper cells, purified T cells were labeled with the Cellvue Lavender Cell Labeling Kit (eBioscience) following the manufacturer's protocol before mixing with T cell-depleted human PBMC. The resulting reconstituted PBMCs were cultured 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. The release of IFNγ and TNFα in culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D System).

As shown in Figures 10A-10B, significant T cell activation (via 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 . Cytokine release for IFNγ in the presence and absence of SKOV-3 tumor cells (Figures 11A-11B) and TNFα (Figures 11C-11D) in the presence and absence of SKOV-3 tumor cells assay, similar correlations were observed.

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

Table 21 - Summary of CD3 binding, in vitro cytotoxicity, T cell activation and cytokine release of various anti-CD3 Fab-HK129-folate low affinity variants.

Based on the data set, three low-affinity variants Fab9, Fab10, and Fab21 as well as the parent molecule Fab1 were selected for detailed in vitro characterization, including in vivo testing in mice. Table 22 shows the comparison between these variants with respect to cytotoxicity and production of both cytokines. As shown, the EC50 for cytokine production is much higher than the EC50 for T cell activation and killing. Therefore, it is possible to identify a range of anti-CD3 Fab concentrations in which cytokine release does not pose major safety concerns, but killing potency and T cell activation are not compromised. This anti-CD3 antibody affinity fine-tuning approach may allow for the decoupling of these two opposing events and achieve a better safety profile without compromising efficacy.

Example 16 :

In-silico immunogenicity analysis of anti-CD3 Fab1, 9 and 10: To assess potential immunogenicity, based on Lonza's Epibase platform, the "HLA Class II-Global Version 4.0" setting was used (Lonza, UK), used computer analogy to scan the amino acid sequences of anti-CD3 Fab1, Fab9 and Fab10 for putative human leukocyte antigen (HLA) class II restricted epitopes (also known as T helper (Th) cell epitopes) The presence. All possible 10-mer peptides derived from the target sequence were analyzed for HLA binding specificity. 43 DRB1, 8 DRB3/4/5, 22 DQ and 12 DP (i.e. a total of 85 HLA class II allotypes) were analyzed at the allotypic level. 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 (number of epitopes) corresponding to the DRB1, DRB3/4/5, DQ and DP genes. As with the humoral response to antigen, the observed Th cell activation/proliferation is often explained in terms of DRB1 specificity. The results in Table 23 indicate that Fab 1, Fab9 and Fab10 correspond to strong potential DRB1 binders 13, 11 and 13 respectively. Table 24 shows that the DRB1 risk scores for each of these 3 Fabs in the global population are comparable to humanized therapeutic antibodies. Among the three Fabs, Fab9 has the lowest immunogenicity.

Example 17 :

The design and synthesis of bi-functional PEG-Folate linkers is shown in Figures 12A-12F. This example illustrates the synthetic routes and structures of various PEG-folate linker compounds.

This example illustrates the synthetic route used to synthesize compound 10.

N ¹⁰ -trifluoroacetylpteroic acid (2) . To 1.0 g of pteroic acid (1) in a round-bottomed flask, 10 ml of trifluoroacetic anhydride was introduced dropwise for 10 minutes under nitrogen. The reaction mixture was stirred at room temperature for 24 hours in the dark. The dark brown solution was filtered through a pad of celite and evaporated. The obtained viscous brown oil was wet-triturated with diethyl ether, and the separated precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum overnight to obtain a crude intermediate in the form of a light brown powder. The crude chelated 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 diethyl ether, and the precipitate was collected by filtration, washed with diethyl ether and dried under vacuum overnight to obtain N10-trifluoroacetylpteroic acid (2) (1.45g crude material) as a light brown powder, which was Further purification was used in the next reaction.

N ¹⁰ -trifluoroacetylpteroate OSu ester (3) . A solution of N10-trifluoroacetylpteroic acid (2) in anhydrous DMSO was treated with N-hydroxysuccinimide (0.43g) at room temperature, followed by EDCI-HCl (2.04g) in one portion . 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 obtain compound (3) as brown powder, MS (ESI) m/z 506 (M+H)+.

Fmoc-Glu-OtBu-Lys(Boc)-OtBu(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 at room temperature overnight, then the solid was filtered off and washed with THF. The combined filtrates were evaporated to dryness and dried under vacuum to give crude Fmoc-Glu(OSu)-OtBu(5) as a white foam (5.3 g). This material was redissolved in THF (20 mL) and added to H-Lys(Boc)-OtBu-HCl (6) (3.39 g) and DIPEA (3.5 mL) in anhydrous THF (50 mL) at room temperature. in the mixture. 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 in vacuo. 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 in vacuo, the residue was triturated with 5% ether/hexane (50 mL). The separated white solid product was filtered, washed with hexane, and dried under vacuum to obtain Fmoc-Glu-OtBu-Lys(Boc)-OtBu(7).

H-Glu-OtBu-Lys(Boc)-OtBu(8) . A solution of Fmoc-Glu-OtBu-Lys(Boc)-OtBu(7) in DCM was treated with diethylamine. The resulting solution was stirred at room temperature for 4 hours until deprotection was judged complete by HPLC analysis. All solvents were removed in vacuo and the residue was purified by silica column chromatography eluting first with dichloromethane and then with 5-10% MeOH in dichloromethane to afford H-Glu-OtBu-Lys (Boc )-OtBu(8).

Compound 9: A solution of amine 8 in anhydrous DMF was treated with N10-trifluoroacetylpteroate OSu ester (3) in one portion. The resulting mixture was stirred at room temperature for 24 hours while monitoring 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 filtrate was evaporated to dryness, redissolved in ethyl acetate, and washed successively with 10% aqueous citric acid solution, water, saturated NaHCO3, and brine. The extracts were dried over sodium sulfate, evaporated and dried under vacuum overnight to give crude material 9 as a brown solid.

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 overall deprotection was judged to be complete by HPLC analysis. All solvent was removed under vacuum and the remaining brown oil was triturated with diethyl ether and sonicated briefly. The separated light-colored precipitate was collected by filtration, washed thoroughly with diethyl ether and dried under vacuum for one day to obtain product 10 as a light yellow powder.

This example illustrates the synthetic route used to synthesize compound 13.

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

Compound 13: To a solution of compound 12 (0.31 g) in DMF (1.5 mL) was added hydrazine at room temperature. H2O (0.19mL). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (~2 mL) and purified by C18 reversed phase HPLC using a 20-90% acetonitrile/0.05% TFA gradient as eluent to obtain compound 13 as a yellow solid after lyophilization. MS(ESI)m/z 831(M+H)+.

The present invention includes linker synthesis as exemplified below by showing the synthetic route for the synthesis of compound 11 (CAS#: 1415328-95-8).

Compound 16: To a solution of tetraethylene glycol 14 in anhydrous THF at room temperature, add small pieces of sodium and stir until completely dissolved. Acrylate 15 was slowly added to the resulting solution over a period of 15 minutes. The reaction mixture was stirred at room temperature for 20 hours, then concentrated in vacuo and resuspended in brine, followed by extraction with ethyl acetate. The combined organic phases were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to afford compound 16 as a clear pale yellow oil.

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 removal of the solvent, the residue was purified on silica gel to afford tosylate 17 as a clear colorless oil.

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

Compound 19: t -Butyl ester 18 was treated with a 1:1 mixture of TFA and DCM at room temperature. After 3 hours, the solvent was removed in vacuo and the residue was taken up in dichloromethane, washed well with brine and dried over sodium sulfate. After removal of the solvent under vacuum, crude carboxylic acid 19 was obtained as a pale yellow clear oil.

Compound 11: Crude carboxylic acid 19 in anhydrous THF was treated with N-hydroxysuccinimide followed by DCC at room temperature. Stirring was continued for 6 hours and the solid was removed by filtration and washed with THF. The filtrate was evaporated and the residue was passed through a pad of silica and washed with EtOAc to afford compound 11 as a colorless oil, which slowly solidified to a white solid on storage.

This example discloses the synthesis of branch joints. For example, the synthetic route used to synthesize compound 25:

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) at 23°C was added DIEA (1.03 mL). After 10 minutes, LCMS showed the reaction was complete. The mixture was washed with IN HCl (50 ml), saturated sodium bicarbonate (50 ml) and brine (50 ml). The organic layer was dried over MgSO4. Removal of the solvent afforded crude acid 21 as a white solid, which was used in the next step without purification.

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

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 by 5% citric acid (20 ml) and brine (50 ml). Dry the organic layer 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)+.

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

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

This example discloses the synthesis of branched PEG-folate compound 30.

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

Compound 27: DCM (3 ml) and TFA (2 ml) were added to compound 26 (0.25 g) at 23°C, and then stirred for 30 minutes. Solvent was removed in vacuo. Dissolve the residue in DCM (~5 ml) and drop the solution into 45 ml MTBE in a conical tube. The precipitate was separated by centrifugation (4000 rpm, 5 min) and dried to give compound 27 as a brown solid; MS (ESI) m/z 1434 (M+H)+.

Compound 29A: To a solution of compound 27 (0.054g) and compound 28A (PEG5K-C5-NHS, 0.17g) in DMF (2ml) at 23°C was added DIEA (0.034mL). stir After stirring for 5 hours, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 minutes, 4000 rpm) to separate the precipitate. Add 45 mL of MTBE to the pellet and centrifuge (5 minutes, 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.

Compound 30A: To a solution of compound 29A (0.24g) in water (10ml) was added hydrazine at 23°C. H2O (0.033mL). After stirring for 24 hours, the mixture was purified by preparative LC using a C18 column using an aqueous gradient of 20% to 100% ACN and 0.05% TFA for 20 minutes. 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 e.g. Figure 12).

Compound 29B: To a solution of compound 27 (0.04g) and compound 28B (PEG10K-C5-NHS, 0.26g) in DMF (6ml) at 23°C was added DIEA (0.040mL). After stirring for 16 hours, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 minutes, 4000 rpm) to separate the precipitate. Add 45 mL of MTBE to the pellet and centrifuge (5 minutes, 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.

Compound 30B: To a solution of compound 29B (0.47g) in water (6ml) was added hydrazine at 23°C. H2O (0.080mL). After stirring for 6 hours, the mixture was purified by preparative LC using a C18 column using an aqueous gradient of 20% to 100% ACN and 0.05% TFA for 20 minutes. 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.

Compound 29C: To a solution of compound 27 (0.040 g) and compound 28C (PEG20K-C5-NHS, 0.51 g) in DMF (8 ml) at 23°C was added DIEA (0.040 mL). After stirring for 16 hours, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 minutes, 4000 rpm) to separate the precipitate. Add 45 mL of MTBE to the pellet and centrifuge 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.

Compound 30C: To a solution of compound 29C (0.67g, <0.031mmol) in water (8ml) was added hydrazine at 23°C. H2O(0.060mL). After stirring for 6 hours, the mixture was purified by preparative LC using a C18 column using an aqueous gradient of 20% to 100% ACN and 0.05% TFA for 20 minutes. 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.

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 hours, the mixture was dropped onto 40 mL of MTBE and centrifuged (5 minutes, 4000 rpm) to separate the precipitate. Add 45 mL of MTBE to the pellet and centrifuge (5 minutes, 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.

Compound 30D: To a solution of compound 29D (0.45g, <0.021mmol) in water (8ml) was added hydrazine at 23°C. H2O (0.080mL). After stirring for 48 hours, the mixture was purified by preparative LC using a C18 column using an aqueous gradient of 20% to 100% ACN and 0.05% TFA for 20 minutes. 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.

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 to 1 mL with water and purified through a desalting column. The collected fraction was lyophilized to obtain compound 28E as a white solid.

Compound 30E: To a solution of compound 28E (0.04g), DMTMMT (0.003g) and DIEA (0.005mL) in DMF (2mL) was added compound 27 (0.009g) at 23°C. Stir After 1 hour, LCMS showed the reaction was complete. To this mixture (crude compound 29E) was added hydrazine in situ. H2O(2ul). After stirring for 1 hour, the mixture was purified by preparative LC using a C18 column using an aqueous gradient of 20% to 100% ACN and 0.05% TFA for 20 minutes. 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.

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

Example 18 :

This example illustrates the construction, expression and purification of bis-amber-containing humanized anti-CD3 Fab lead molecules. Adding a second pAF incorporation site at the anti-CD3 Fab light chain position LL157 in Fab1-HK129pAF, Fab9-HK129pAF and Fab10-HK129pAF generates new Fab molecules such as Fab1-HK129pAF-LL157pAF, Fab9-HK129pAF-LL157pAF, respectively. Fab10-HK129pAF-LL157pAF. This allows conjugation of two folate plus two 5KPEG molecules in each Fab using any bifunctional linker as described in the previous examples.

CD3-PEG-folate purified protein for in vitro activity, in vivo efficacy and PK studies was analyzed by SDS gel electrophoresis (SDS PAGE). Purified CD3 was conjugated with 5K or 10K PEG-folate at the pAF position using oxime chemistry, followed by post-conjugation purification using cation exchange chromatography to separate the unconjugated, single-position and dual-position conjugated forms (Figures 13A-13B ). After purification, the composition was formulated in 50mM histidine, 100mM NaCl, 5% trehalose pH 6 and sterile filtered.

Figure 13A shows 5 μg of each purified CD3-folate bispecific antibody conjugated to 5K PEG. Lanes 3 and 6 represent unconjugated CD3 Fab compositions incorporating single and double pAF, respectively. Lanes 4 and 7 represent CD3 Fab compositions with single and double pAF and folate, respectively. Lanes 5 and 8 represent conjugated 5KPEG-folate and bis-5KPEG-bisfolate, respectively.

Figure 13B shows the SDS-PAGE results of loading 10 μg of protein per well under non-reducing conditions. Lanes 2 and 7 represent unconjugated CD3 Fab compositions, lanes 3 and 4 each represent a different bis-conjugated bis 5KPEG-bisfolate CD3 Fab composition, and lanes 5 and 6 each represent bis-conjugated bis 10KPEG-bisfolate CD3 Fab compositions and 10KPEG-folic acid. The data (Figure 13A-B) show high purity (>90%) for all samples, with the expected increase in molecular weight based on PEG size.

To increase conjugation efficiency, the purified CD3 Fab composition was conjugated to PEG-folate using a two-step conjugation method. Folate was conjugated at the pAF position using oxime chemistry, followed by PEG conjugation using maleimide-thiol click chemistry with 5K, 10K or 20K PEG at the C-terminal cysteine of both heavy and light chains . Figure 13C shows SDS-PAGE loading 10 μg of protein per well under non-reducing (lanes 2-5) and reducing (lanes 7-10) conditions. Lanes 2 and 7 represent the unconjugated composition of difolate, and lanes 3 and 8, 4 and 9, and 5 and 10 each represent the difolate-C-terminal bis-5KPEG, bis-10KPEG, and bis-20KPEG compositions, respectively. The data in Figure 13C shows high purity (>95%) for all samples, with the expected increase in molecular weight based on PEG size.

Example 19 :

This example demonstrates the effect of bisfolate and bisPEG conjugation on the composition of CD3 Fab1 and two low affinity variants Fab9 and Fab10. Human CD3 Binding and Cytotoxicity: Table 25 shows the effect of various modifications, including the conjugation of bis 5KPEG-bisfolate in the presence of 50 nM SFA, on the binding affinity and cytotoxicity of Fab1 molecules.

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

Table 27 compares the binding affinities of the parent Fab1 and two low affinity variants, Fab 9 and 10, to human CD3 in the presence of 20 nM SFA. As shown, conjugation of bis 5K PEG-bisfolate, accomplished using a PEG-folate bifunctional linker as described in the Examples above, resulted in reduced human CD3 binding for all 3 Fabs compared to their unconjugated controls and corresponding cells. Reduced toxicity potential.

T cell activation and cytokine release: Table 27 shows the activation of the T cell marker CD69 by 3 modified Fabs and the release of the two cytokines IFNγ and TNFα. As shown in the examples above, a general correlation between CD3 binding strength, cytotoxic potential, T cell activation and subsequent cytokine release was found across all Fabs even after significant modification by bisfolate-bis 5KPEG conjugation. Still holds true. This suggests that by simultaneously reducing CD3 affinity and increasing tumor-associated antigen (TAA) affinity, efficacy can be preserved while minimizing toxicity due to cytokine release syndrome. Additionally, improved PK properties, including half-life (T1/2) extension, are achieved through site-specific PEGylation using proprietary UAA (unnatural amino acid) incorporation technology.

Example 20 :

In vitro cytotoxicity data showed that the CD3-folate bispecific antibody selectively killed KB cells expressing FOLRα. KB cells were treated with increasing concentrations of CD3-folate bispecific antibody in the presence of 50 nM folate (physiologically relevant concentration of folate). The most potent CD3-folate bispecific antibody, Fab1-HK129-LL157-bifolate, containing two folate molecules, had an IC50 value of 1.3 pM (Figure 14). The CD3-folate bispecific antibody Fab1-HK129-folate containing monofolate showed an IC50 value of 40.3 pM and was 31-fold less potent than Fab1-HK129-LL157-bifolate. This data shows that two folates increase the potency of CD3-folate bispecific antibodies. The addition of 5KPEG reduces effectiveness. A 4.9-fold reduction in potency was observed between the single CD3-folate bispecific antibody Fab1-HK129-folate and Fab1-HK129-5KPEG-folate with an IC50 value of 198 pM. A 37.3-fold decrease in potency was observed between Fab1-HK129-LL157-bifolate and Fab1-HK129-LL157-bis5KPEG-bifolate with an IC50 value of 48.5 pM. These data demonstrate that CD3-folate bispecific antibodies maintain potent in vitro cytotoxicity at physiologically relevant concentrations of folate.

Example 21 :

In vitro cytotoxicity data showed that the CD3-folate bispecific antibody selectively killed FOLRα-expressing SKOV3 cells in the presence of 20 or 50 nM folic acid. SKOV3 cells were treated with increasing concentrations of CD3-folate bispecific antibody in 20 or 50 nM folate (physiologically relevant concentrations of folate) (Figures 15A and 15B). The CD3-folate bispecific antibody showed a decrease in potency, 5.6-fold to 8-fold, when the folate concentration was increased from 20 nM to 50 nM (near the maximum normal physiologically relevant concentration of folate). The addition of 5KPEG also resulted in reduced potency of the CD3-folate bispecific antibody. A 5.4-fold reduction was observed between monofolate and mono-5KPEG folate bispecific antibodies, and a 22.9-fold reduction between bifolate and bi-5KPEG folate bispecific antibodies. No significant difference in potency was observed between single-PEGylated CD3-folate bispecific antibodies and double-PEGylated CD3-folate bispecific antibodies. The data in Table 28 demonstrate that CD3-folate bispecific antibodies can kill in the presence of physiologically relevant concentrations of folate. CD3-folate bispecific antibodies can kill FOLRα-expressing cells, although pegylated antibodies are less effective than non-PEGylated antibodies.

Example 22 :

Further in vitro cytotoxicity studies were performed in the presence of 20 or 50 nM 5-mTHF. SKOV3 was treated with increasing concentrations of CD3-folate bispecific antibody in the presence of 5-methyltetrahydrofolate (5-mTHF) at 20 or 50 nM (physiologically relevant concentrations of the major form of folate found in human serum). cells (Figures 16A and 16B). 5-mTHF has a binding affinity to FOLRα of 1-10 nM and does not bind to FOLRα as well as folic acid, which has a binding affinity of less than 1 nM. The CD3-folate bispecific antibody maintained very potent IC50 values ranging between 0.03 to 0.16pM and 0.1 to 1.5pM for dual CD3-folate bispecific antibodies and single CD3-folate bispecific antibodies, respectively. The data in Table 29 demonstrate that the CD3-folate bispecific antibody has potent in vitro cytotoxicity against FOLRα-expressing SKOV3 cells in the presence of physiologically relevant concentrations of 5-mTHF.

Example 23 :

Mouse pharmacokinetic studies were performed in CD1 mice: CD3-folate bispecific antibodies were injected intravenously via the mouse tail vein of CD-1 mice at a dose of 1 or 5 mg/kg. Blood samples were collected at nine time points and analyzed via ELISA. The data clearly show that the addition of 5KPEG increases serum exposure (AUClast) (Tables 30-31 and Figure 17). Fab1-HK129-5KPEG-folate at 1 mg/kg showed a 4.3-fold increase over Fab1-HK129-folate, while Fab1-HK129-5KPEG-folate at 5 mg/kg showed a 5-fold increase over Fab1-HK129-folate. Fab1-HK129-LL157-bis 5KPEG-bisfolate showed a 16.25-fold improvement over Fab1-HK129-LL157-bisfolate at 1 mg/kg, while Fab1-HK129-LL157-bis 5KPEG-bisfolate showed a 16.25-fold improvement over Fab1 at 5 mg/kg. -HK129-LL157-Difolate increased by 21.7 times. Data show that there is a 3.9-fold difference between Fab1-HK129-5KPEG-folic acid and Fab1-HK129-LL157-bis 5KPEG-bisfolate. An improvement in serum half-life (T1/2) was also observed when 5KPEG was added to the CD3-folate bispecific antibody. The greatest improvement in serum half-life was observed with the incorporation of two 5KPEGs into the CD3-folate bispecific antibody. Fab1-HK129-LL157-bis5KPEG-bisfolic acid showed a 4.2-fold increase in serum half-life compared to Fab1-HK129-LL157-bisfolic acid at 1 mg/kg, while Fab1-HK129-LL157-bis5KPEG-bisfolic acid at 5 mg/kg Folic acid showed a 6.25-fold increase in serum half-life compared to Fab1-HK129-LL157-bifolate. Data show that the addition of 5KPEG improves serum exposure and serum half-life of CD3-folate bispecific antibodies, which results in less frequent dosing to achieve effective serum exposure in vivo.

Example 24

This example illustrates the killing of human M2 macrophages by a CD3-folate-folate bispecific antibody: macrophages are a heterogeneous population of cells that play a role in host defense. Classically activated macrophages (M1 macrophages) have pro-inflammatory functions, recruit tumor-infiltrating lymphocytes, and have anti-tumor activity, whereas M2 macrophages have anti-inflammatory effects and participate in tissue remodeling, cancer cell migration, invasion, and metastasis. Human M2 Macrophage The cells are associated with cancer cell proliferation and are associated with poor prognosis in ovarian cancer. Inhibiting M2 macrophages enhances the activity of immuno-oncology therapies such as checkpoint inhibitors. Human M2 macrophages express FOLRβ or FRβ, which or FR has similar folate binding affinity to FOLRα or FRα. Therefore, strategies to increase the ratio of M1 to M2 macrophages by repolarizing M1 macrophages or by selectively killing M2 macrophages offer potential therapeutic approaches for cancer treatment.

Study 1: To evaluate the effect of CD3-folate bispecific antibodies on macrophages, the following experiments were performed: Fab1-HK129-LL157-pAF, Fab1-HK129-LL157-folate, and Fab1-HK129-LL157-5KPEG folate were combined with Human M2 macrophages and human T cells were cultured together in the presence of 50 nM folic acid. Data show that Fab1-HK129-LL157-folic acid and Fab1-HK129-LL157-5KPEG-folic acid kill human M2 macrophages, and their IC50 values are 1.2pM and 112.1pM respectively (data not shown). Fab1-HK129-LL157-pAF lacking folate does not result in human M2 macrophage killing. Data support that the cytotoxicity of Fab1-HK129-LL157-folate and Fab1-HK129-LL157-5KPEG-folate is specific for binding to FOLRβ. In addition, data indicate that in addition to killing tumor cells expressing FOLRα, CD3-folate bispecific antibodies can also kill M2 macrophages and possibly other immunosuppressive cells expressing FOLRα/β.

Study 2: In these studies, monocyte-derived macrophages were generated from human blood from healthy donors and treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) for M1 macrophages Differentiate or treat with macrophage colony-stimulating factor (M-CSF) for M2 macrophage differentiation.

Prior to conducting the study, folate receptor beta (FOLRβ or FR-β) expression was initially measured by flow cytometry and found that M2 macrophages were thinner than M1 macrophages in terms of median fluorescence intensity (MFI). increased by 26 times.

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

Effect of PEGylation on CD3-folate bispecific antibodies: Table 32 shows IC50 data for in vitro macrophage cytotoxicity of monofolate-containing CD3-folate bispecific antibodies in the presence of 20 nM folate. The average IC50 of Fab1-HK129-folate in M1 macrophages was 85.0 pM (range 53.5-120.0 pM) and in M2 macrophages was 5.6 pM (range 1.4 pM-15.0 pM). Based on the IC50 ratio, M2 macrophages were on average 26-fold (range 5-42-fold) better than M1 using Fab1-HK129-folate. Fab1-HK129-5KPEG-folate showed an average IC50 of 616.8pM (range 198.0-908.9pM) in M1 macrophages and an average IC50 of 13.4pM (range 2.3-33.2pM) in M2 macrophages, where The increased mean IC50 ratio between M1 and M2 macrophages was on average 69-fold greater in M2 macrophages (range 23-126-fold). These results indicate that the monofolate-containing CD3-folate bispecific antibody is specific for M2 macrophage killing and that the PEGylation of the monofolate-containing CD3-folate bispecific antibody (Fab1-HK129-5KPEG-folate ) is more selective for killing M2 macrophages than Fab1-HK129-folic acid.

In vitro macrophage cytotoxicity results of monofolate-containing CD3-folate bispecific antibodies from a representative donor, donor 6007, are depicted in Figure 18A. Arrows and numbers indicate fold differences in IC50 between M1 and M2 macrophages. The dashed lines represent Fab1-HK129-folate in M1 and M2, while the solid lines represent Fab1-HK129-5KPEG-folate in M1 and M2.

Table 33 shows IC50 data for the cytotoxicity of bifolate-containing CD3-folate bispecific antibodies to macrophages in vitro in the presence of 20 nM folate. Fab1-HK129-LL157-bifolate had an average IC50 of 29.2pM (range 10.9-42.8pM) in M1 macrophages and an average IC50 of 1.5pM (range 0.1pM-5.5pM) in M2 macrophages, where The average IC50 ratio between M1 and M2 macrophages differed 72-fold (range 6-212-fold). Fab1-HK129-LL157-bifolate-bis 5KPEG showed an average IC50 of 1620.8 pM (range 834.7-2651 pM) in M1 macrophages and an average IC50 of 15.7 pM (range 3.6-52.4 pM) in M2 macrophages. , where the average IC50 ratio between M1 and M2 macrophages improved 181-fold in M2 macrophages (range 49-501-fold). These results indicate that bifolate-containing CD3-folate bispecific antibodies are specific for killing M2 macrophages. Although PEGylation of bifolate-containing CD3-folate bispecific antibodies (Fab1-HK129-LL157-bifolate-bis5KPEG) was shown to be less potent, it was more selective for M2 macrophage killing than Fab1- HK129-LL157-bifolate or Fab1-HK129-5KPEG-folate, as shown in Table XXX.

Figure 18B shows the cytotoxicity results of bifolate-containing CD3-folate bispecific antibodies on macrophages in vitro from a representative donor, Donor 6007. Arrows and numbers indicate fold differences in IC50 between M1 and M2 macrophages. The dashed line represents Fab1-HK129-LL157-bisfolate in M1 and M2, and the solid line represents Fab1-HK129-LL157-bisfolate-bis5KPEG in M1 and M2.

Further studies were performed to simulate physiologically relevant concentrations of the major folate forms present in human serum, which range between 9.1-45.1 nM. Within this physiological range, 86.7% (37.5 nM) is the major folate metabolite 5-methyl-tetrahydrofolate (5-mTHF), while only 4% (1.2 nM) is unmetabolized folate (Pfeiffer et al., Br .J. Nutr., June 28, 2015: 113(12):1965-1977). As is well known in the art, the binding affinity of folic acid to FR-β is less than 1 nM, while the affinity of 5-mTHF to FR-β is 1-10 nM. Based on this information, it was hypothesized that the concentration or composition of folate and 5-mTHF could affect the activity of the CD3-folate bispecific antibody. To assess this, the following experiments were performed: In vitro macrophage cytotoxicity data in the presence of 45 nM 5-mTHF are shown in Table 34 and Table 35. In M1 or M2 macrophages, the average IC50 of Fab1-HK129-folate was 9.1 pM and 0.31 pM, respectively, and the average IC50 of Fab1-HK129-5KPEG-folate was 48.5 pM and 1.26 pM, respectively. The IC50 of Fab1-HK129-LL157-bisfolate in M1 averaged 4.0pM and the IC50 in M2 averaged 0.05pM, while the IC50 of Fab1-HK129-LL157-bisfolate-bis5KPEG in M1 averaged 99.4pM and The average IC50 in M2 is 0.85pM. These data indicate that the CD3-folate bispecific antibody is more effective in the presence of 5-mTHF than in the presence of folic acid (Table 32 and Table 33). The IC50 ratios between M1 and M2 of monofolate-containing CD3-folate bispecific antibodies averaged 43-fold and 57-fold, and the IC50 ratios between M1 and M2 of bifolate-containing CD3-folate bispecific antibodies averaged 43-fold and 57-fold are 89 times and 212 times. These data demonstrate that CD3-folate-containing bispecific antibodies maintain specific killing of M2 macrophages and that pegylation provides greater specificity at physiologically relevant 5-mTHF concentrations.

Overall, the data indicate that doubly PEGylated CD3-folate compositions exhibit greater efficacy on M2 macrophages compared to singly PEGylated CD3-folate compositions in the presence of folate or 5mTHF metabolites. Specificity of killing. Therefore, an increase in selectivity was observed with more PEGylation.

Further studies with a CD3-folate bispecific antibody were also performed in human myeloid derived suppressor cells (MDSC) to study FRβ expression. Human peripheral blood mononuclear cells (PBMC) from healthy donors were treated with Fab1-HK129-5KPEG-folate and Fab1-LL157-bis5KPEG-bifolate to test their targeting against mononuclear MDSCs (mMDSCs). Cytotoxic activity at 0, 1, 10 and 100 pM, for each CD3-folate bispecific antibody, was added to PBMC and cultured in a 37°C and 5% CO2 incubator for 24 hours. After culture, the mMDSCs were gated by the CD3-/CD33+/CD11b+/CD14+/HLA-DR low population using flow cytometry and the percentage (%) of viable cells relative to untreated controls was measured.

Based on the observations, the percentage of mMDSCs decreased in a dose-dependent manner. Fab1-HK129-5KPEG-folate showed a dose-dependent reduction from 72% to 24% at 10 pM relative to 100 pM, respectively. Similarly, Fab1-HK129-LL157-bis5KPEG-bifolate showed a dose-dependent reduction from 88% to 22% at 10 pM relative to 100 pM, respectively. These results indicate that treatment with a CD3-folate bispecific antibody selectively eliminates mMDSCs as long as non-MDSCs with high HLA-DR are retained.

Given that these suppressor cells can express folate receptors and participate in the inhibition of immuno-oncology drug activity, it is suggested that combination therapies using checkpoint inhibitors and other immuno-oncology drugs could be used to target various cancers or conditions in which folate receptors are expressed /Treatments for diseases/disorders. This supports the use of the CD3-folate compositions of the invention as a valuable therapeutic agent that is significantly different from other immuno-oncology therapies.

Example 25 :

This example illustrates the binding affinity of CD3-folate bispecific antibodies for FOLRα in several cell types. Binding affinity of CD3-folate bispecific antibody 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. KB cells bound, and Fab1-HK129-5KPEG-folate bound to FOLRα-expressing KB cells with an affinity of 3.69 nM. Fab1-HK129-LL157-bifolate binds to KB cells with an affinity of 0.85 nM, while Fab1-HK129-LL157-bis 5KPEG-bifolate binds to KB cells with an affinity of 2.28 nM. Pegylated antibodies retained similar binding affinity to FOLRα-expressing KB cells as non-PEGylated antibodies, which was in the low nM range.

Binding affinity of CD3-folate bispecific antibodies to human T cells: Fab1-HK129-pAF (anti-CD3 Fab) does not contain folate or PEG and binds to human T cells with an affinity of 1.59 nM (Table 37). Fab1-HK129-folate binds to CD3-expressing human T cells with an affinity of 1.53 nM, while Fab1-HK129-5KPEG-folate binds to CD3-expressing 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 0.604nM. Fab1-HK129-LL157-bifolate binds to human T cells with an affinity of 0.669 nM, while Fab1-HK129-LL157-bis 5KPEG-bifolate has an affinity of 3.34 nM. These data indicate that the binding affinity of pegylated CD3-folate bispecific antibodies to human T cells is approximately 5-fold lower than that of non-PEGylated antibodies. These data also indicate that the CD3-folate bispecific antibody has low nM binding affinity for human T cells.

Binding affinity of CD3-folate bispecific antibody 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 bound to CD3-expressing cynomolgus monkey T cells with an affinity of 2.69 nM, while Fab1-HK129-5KPEG-folate bound with an affinity of 3.31 nM. This suggests that CD3-folate bispecific antibodies with a single amber position have similar binding affinities to cynomolgus monkey T cells. CD3-folate constructs with double amber positions showed similar binding affinities in cynomolgus monkey T cells. As shown in Table 38, Fab1-HK129-LL157-pAF bound to cynomolgus monkey T cells with an affinity of 0.995 nM. Fab1-HK129-LL157-bifolate bound to cynomolgus monkey T cells with an affinity of 1.0 nM, while Fab1-HK129-LL157-bis 5KPEG-bifolate bound to cynomolgus T cells with an affinity of 5.01 nM. Therefore, the data further indicate that the pegylated CD3-folate bispecific antibody binds to cynomolgus monkey T cells with approximately 5-fold lower affinity than the non-PEGylated CD3-folate bispecific antibody. Overall, the data indicate that the CD3-folate bispecific antibody has low nM binding affinity to cynomolgus monkey T cells.

Example 26 :

The following study illustrates in vivo safety and efficacy studies in mice using the CD3 folate antibodies of the invention.

Study 1: The anti-tumor efficacy of an anti-CD3-folate bispecific antibody was tested by multiple dose administration in female immunocompromised mice bearing human cervical tumors derived from KB cell lines. Non-targeting anti-CD3 antibody was included as a control. Female NSG mice bearing KB cervical tumors (inoculated with 2.0 × 106 cells from passage 7 and initially grown from frozen passage 3) were divided into 5 groups of 8 animals each. When the tumor size was approximately 100 mm3, 7.5 × 106 PanT cells were inoculated intraperitoneally into mice. After 24 hours, mice were randomly divided into the following treatment groups: G1: anti-CD3 Fab1-HK129-pAF control (0.05mpk), G2 and G3 each with 0.01mpk and 0.05mpk of CD3 Fab1-HK129-LL157-bifolate, respectively. - Bi-5KPEG, G4:Fab9-HK129-LL157-bifolate-bis 5KPEG (0.125mpk), and G5:CD3 Fab1-HK129-folate control (0.25mpk); and tumor volume (Figure 19A) and mass/body weight ( Figure 19B). All mice were dosed intravenously (IV) on day 7 when tumors averaged approximately ~125 mm3. Animals were monitored for tumor growth (measured by calipers) and body weight twice weekly. As shown in Figure 19A, all CD3-folate bispecific compositions showed efficacy, with group 3 (Fab1-HK129-LL157-bifolate-bis5KPEG, 0.05mpk) having the greatest impact on hindering tumor growth (TGI =82%). All compositions caused weight loss except the control CD3 Fab-HK129-pAF antibody (Figure 19B).

Biomarker analysis: Blood samples were drawn from each animal before starting treatment and on days 7 and 24 after the start of treatment. Samples were analyzed for the percentage of human CD45/mouse CD45 by FAC analysis to determine whether treatment increased the peripheral human T cell population as proposed by CD3-folate activation/targeting. At the end of the study, tumors were analyzed for the presence of tumor-infiltrating lymphocytes (TILs) by FAC using the human lymphocyte marker hCD45. All CD3-folate treatment groups showed anti-tumor efficacy with varying degrees of ability to increase human CD45 blood levels, with human CD45 having the highest levels and attributable to 0.05 mpk of the bispecific antibody Fab1-HK129-LL157-bi Folic acid-bis 5KPEG treatment (Figure 19C). This study also examined tumor growth inhibition and the present invention's The ability of CD3-folate compositions to promote tumor-infiltrating lymphocytes (TILs). Additionally, the presence of TILs, a marker of tumor immune surveillance activation, was found to be increased in all treatment groups compared to the control group, with group 3 (Fab1-HK129-LL157-bisfolate-bis5KPEG, 0.05mpk) having Highest TIL induction (Figure 19D).

Study 2: This study examined tumor growth inhibition (TGI) and changes in human CD45 percentage in mouse blood/tumor infiltrate in NCG mice bearing KB tumors. Single (Fab1-HK129-LL157-folate-5KPEG) and bis (Fab1-HK129-LL157-bifolate- Antitumor efficacy of bi-5KPEG) pegylated anti-CD3-folate bispecific antibodies. Non-targeting CD3 antibody was included as a control. Thirty-five female NCG mice bearing KB cervical tumors (inoculated with 2.0 × 106 cells from passage 5 and initially grown from frozen passage 3) were divided into 3 groups of 10 animals each. When tumors were approximately 85-100 mm3, mice were inoculated intraperitoneally with 6.0×106 PanT cells. After 24 hours, the mice were randomly divided into the following treatment groups: G1: CD3 Fab1-HK129-pAF control, G2: Fab1-HK129-LL157-folate-5KPEG, G3: Fab1-HK129-LL157-bisfolate-bis5KPEG, 0.025mpk every 5 days per group (Figures 20A and 20B). On day 7, all mice were inoculated with 6.0 × 106 Pan-T cells and administered intravenously (IV) on day 8, when tumors averaged approximately ~100 mm3. Animals were monitored twice weekly for tumor growth (Figure 20A) (measured by calipers) and body weight (Figure 20B). All CD3-folate targeting compositions showed varying degrees of efficacy, among which G2: Fab1-HK129-LL157-folate-5KPEG (single PEG composition) had the greatest impact on hindering tumor growth (TGI=85%), and in 5 out of 10 mice induced complete tumor regression (CR), compared to G3:Fab1-HK129-LL157-bisfolate-bis-5KPEG (bis-PEG composition), which had a TGI of 76% and 10 Three of the animals had CR (Fig. 20A). All tested were tolerated, with G3 (dual PEG composition) showing slight weight loss in several mice, but these weight losses did not exceed 15% (Figure 20B).

Biomarker analysis: Blood samples were drawn from each animal on days 7 and 14 after the start of the above treatments. Blood samples were analyzed for percent changes in human CD45 to determine whether treatment increased peripheral human T cell populations through CD3-folate activation/targeting. At the end of the study, tumors were analyzed for tumor-infiltrating lymphocytes (TILs) by FAC using the human lymphocyte marker hCD45. All CD3-folate treatment groups demonstrated anti-tumor efficacy and had varying degrees of ability to increase human CD34 levels in blood (Fig. 20C) and tumors (Fig. 20D), with the highest levels of human CD45 and TGI attributable to treatment with single Pegylated CD3-folate targeted composition therapy. Study 3: showed similar results to Study 1 and Study 2. 50 NSG humanized female mice (CD34+ from Jackson laboratories) bearing KB cervical tumors (inoculated with 2.0 × 106 cells starting at passage 5 and originally grown from frozen passage 3) were divided into 5 groups, 10 animals per group. Treatment begins when the tumor is approximately 80-100mm3. G1: Fab1-HK129-pAF control (0.0125mpk), G2: Fab1-HK129-bifolate (0.0025mpk), G3: Fab1-HK129-bifolate (0.0125mpk), G4: Fab1-HK129-bifolate-bis 5KPEG (0.0025mpk), G5: Fab1-HK129-bisfolate-bis5KPEG (0.0125mpk). All mice were dosed intravenously (IV) twice weekly. Animals were monitored twice weekly for tumor growth (Figure 21A) (measured by calipers) and body weight (Figure 21B). On day 5 of treatment (the first day after the second dose), T cell activation was analyzed in blood samples drawn from the animals. Blood samples were analyzed for the percentage of human CD45, CD3, CD25 and CD69 by FAC analysis to determine whether the CD3-folate targeting composition increases the expression of T cell activation markers (Figures 21C-21F). Tumor-infiltrating lymphocytes (TILs) were also analyzed at the end of the study, with 5 tumors from each group analyzed for TIL (CD45, CD3, and CD8) by FAC. All CD3-folate-targeting compositions demonstrated varying degrees of efficacy, however, at similar doses, all pegylated CD3-folate-targeting compositions (TGI of 87% in the G4 group and 91% in the G5 group %) were superior to their non-PEGylated isomers at the same dose (TGI of 51% in the G2 group and 67% in the G3 group), including induction of T cell activation markers CD25/CD69, increased TIL, and have greater anti-swelling Tumor growth inhibition. All test substances caused slight weight loss in mice, but none exceeded 15%.

Study 4: Testing the CD3-folate bispecific antibodies Fab1-HK129-LL157-bifolate and Fab1-HK129-LL157 by multiple-dose administration in female immunocompromised mice bearing human cervical tumors derived from KB cell lines - Anti-tumor efficacy of bisfolate-bis 5KPEG. A non-targeting CD3 antibody (Fab1-HK129-pAF) was included as a control. Sixty-five female NSG mice bearing KB cervical tumors (inoculated with 2.0 × 106 cells from passage 7 and initially grown from frozen passage 3) were divided into 6 groups of 10 animals each. When tumors were approximately 85-100 mm3, 7.5×106 PanT cells were inoculated intraperitoneally into mice. After 24 hours, the mice were randomly divided into the following treatment groups: G1: CD3 Fab1-HK129-pAF control, G2: Fab1-HK129-LL157-bisfolate-bis5KPEG (0.0125mpk), G3: Fab1-HK129-LL157- Bifolate-bis 5KPEG (0.05mpk), G4: Fab1-HK129-LL157-bifolate (0.0125mpk), G5: Fab1-HK129-LL157-bifolate (0.05mpk). All mice were dosed intravenously (IV) on day 8 when tumors averaged approximately ~100mm3. Animals were monitored for tumor growth (measured by calipers) and body weight twice weekly. As shown in Figure 22A, all CD3-folate targeting compositions showed anti-tumor activity (TGI=G2: 92%, G4: 55%, G5: 90%), among which the G3 group (Fab1-HK129-LL157-bifolate -Double 5KPEG, 0.05mpk) had the greatest impact on hindering tumor growth (TGI=99%) and resulted in 6 of 8 mice experiencing complete tumor regressions (CR). With the exception of the control CD3 Fab antibody, all test compositions caused some weight loss, but no more than a 15% loss (Figure 22B). The results of this study indicate the existence of a dose dependent response, which can be attributed to the biophysical properties of the addition of PEG to CD3 Fab-folate compounds.

Study 5: Testing the CD3-folate bispecific antibodies Fab1-HK129-LL157-bifolate and Fab1-HK129 by multiple-dose administration in female immunocompromised mice bearing human ovarian tumors derived from the OV-90 cell line - Anti-tumor efficacy of LL157-bisfolate-bis 5KPEG (Figures 23A and 23B). OV-90 was selected to determine whether the CD3 Fab-folic acid compositions of the invention are active in models resistant to platinum-based therapies, the standard of care for ovarian cancer. A non-targeting CD3 antibody (Fab1-HK129-pAF) was included as a control. Seventy female NSG mice bearing OV-90 ovarian tumors (inoculated with 7.5 × 106 cells from passage 5, initially grown from frozen passage 2) were divided into 6 groups of 10 animals each. When tumors were approximately 100-200 mm3, 7.5 × 106 PanT cells were inoculated intraperitoneally into mice. After 24 hours, the mice were randomly divided into the following treatment groups: G1: Fab1-HK129-pAF control, G2: Fab1-HK129-LL157-bifolate (0.0125mpk), G3: Fab1-HK129-LL157-bifolate (0.05 mpk), G4: Fab1-HK129-LL157-bisfolate-bis5KPEG (0.0125mpk), G5: Fab1-HK129-LL157-bisfolate-bis5KPEG (0.05mpk), and G6: carboplatin (100mpk). All mice were dosed intravenously (IV) on day 8 when tumors averaged approximately ~100mm3. Groups 1 to 5 were administered every 5 days for a total of 3 doses, while Group 6 was administered 7 days later (day 15) for a total of 2 doses. Animals were monitored for tumor growth (measured by calipers) and body weight twice weekly. Although the OV-90 model was resistant to the standard of care treatment of ovarian cancer with carboplatin (G6 group), all CD3-Fab folate-targeted compositions tested showed varying degrees of efficacy, with the G5 group (Fab1-HK129-LL157 -bisfolate-bis 5KPEG, 0.05mpk) had the greatest impact on inhibiting tumor growth (TGI=81%); Figure 23A. Except for the control group CD3 Fab antibody, all test compositions caused weight loss, but the weight loss in each group did not exceed 20% (Figure 23B).

Similar to the results of Study 4, Study 5 showed that when administered at the same concentration, the group treated with Fab1-HK129-LL157-bisfolate-bis5KPEG had more Good anti-tumor activity. This can be attributed to the enhanced biophysical properties resulting from the addition of PEG to the CD3 Fab-folate composition. It should also be noted that strong antitumor effects are still observed in ovarian cancer cells with low copy numbers of folate receptors (e.g. ~10K copies of OV-90 per cell). This supports the use of the CD3 Fab-folate compositions of the invention as first-line therapeutics in the treatment of patient populations without the prerequisite of high folate receptor copy number. This is further confirmed by the in vitro cytotoxicity assay of the present invention, showing that cells with low folate receptor copy numbers still have EC50 values in the picomolar range.

Example 27 :

Human clinical trials of the safety and efficacy of anti-CD3 Fab-folate bispecific antibody (Fab-bifolate, Fab-folate-PEG and Fab-bifolate-biPEG) compositions containing non-naturally encoded amino acids.

Objective: To compare the safety and pharmacokinetics of subcutaneously administered anti-CD3 Fab-folate bispecific recombinant human antibodies containing non-naturally encoded amino acids with commercially available products specific for the same target antigen.

Patients: Eighteen healthy volunteers were included in the study, ranging in age from 20 to 40 years and weighing from 60 to 90 kg. Subjects will have no clinically significant abnormal laboratory values in hematology or serum chemistry, as well as negative urine toxicology screens, HIV screens, and hepatitis B surface antigen. They should not have any signs of: high blood pressure; a history of any primary hematological disorder; a history of significant hepatic, renal, cardiovascular, gastrointestinal, genitourinary, metabolic, neurological disease; a history of anemia or seizures; resistance to bacteria or Known susceptibility to products of mammalian origin, PEG, or human serum albumin; habitual and heavy consumer of caffeinated beverages; participation in any other clinical trial or blood transfusion or blood donation within 30 days of study entry; Have been exposed to anti-CD3 antibodies within three months of study; become ill within 7 days of study entry; and have significant abnormalities in pre-study physical examination or clinical laboratory evaluation within 14 days of study entry. Safety was evaluable in all subjects and all blood collections for pharmacokinetic analyzes were collected as planned. All studies were performed with institutional ethics committee approval and patient consent.

This will be a Phase I, single-center, open-label, randomized, two-cycle crossover study in healthy female volunteers. Eighteen subjects were randomly assigned to one of two treatment sequence groups (nine subjects/group). Anti-CD3 antibodies are administered via local or systemic administration periods by any route commonly used to introduce the antibodies to a subject in need thereof by one of skill in the art. The anti-CD3 antibody composition according to the present invention can be administered subcutaneously, intramuscularly, intradermally, intraarticularly, intrapleurally, Administration may be intraperitoneal, intraarterial or intravenous, but the most appropriate route in any given case will depend on the nature and severity of the disease and/or condition being treated. For example, a bolus subcutaneous (s.c.) or intravenous injection using equivalent doses of a bispecific anti-CD3 Fab-folate antibody containing a non-naturally encoded amino acid and the Selected commercially available products. Dosage and application frequency for commercially available products are as directed on the package label. Additional dosing, frequency of dosing, other parameters required, use of commercially available products can be added to the study by including other groups of subjects. Each dosing period was separated by a 14-day washout period. For each of the two dosing periods, subjects were restricted to the study center for at least 12 hours before and 72 hours after dosing, but not between dosing periods. If additional dosing, frequency, or other parameters need to be tested for pegylated bispecific anti-CD3 antibodies, additional groups of subjects may be added. Experimental preparations of anti-CD3 antibodies are bispecific anti-CD3 Fab-folate antibodies containing non-naturally encoded amino acids.

Blood sampling: Blood was drawn continuously by direct venipuncture before and after administration of anti-CD3 Fab-folate bispecific antibody. At approximately 30, 20, and 10 minutes before dosing (3 baseline samples) and at approximately the following times after dosing: 30 minutes and 1, 2, 5, 8, 12, 15, 18, 24, 30, 36, 48, At 60 and 72 hours, venous blood samples (5 mL) were obtained for determination of serum anti-CD3 antibody concentrations. Each serum sample was divided into two aliquots. All serum samples were stored at -20°C. Serum samples were shipped on dry ice. Fasting clinical laboratory tests (hematology, serum chemistry, and urinalysis) were performed immediately before initial dosing on Day 1, on the morning of Day 4, immediately before dosing on Day 16, and on the morning of Day 19.

Bioanalytical methods: Serum bispecific anti-CD3 Fab-folate antibody concentrations were measured using radioimmunoassay (RA) or ELISA kit procedures.

Safety Determination: Record vital signs before each dose (days 1 and 16) and 6, 24, 48 and 72 hours after each dose. Safety determinations were based on the incidence and type of adverse events and changes from baseline in clinical laboratory tests. Additionally, changes from pre-study measurements in vital signs, including blood pressure and physical examination results, were assessed.

Data Analysis: Post-dose serum concentration values were corrected relative to pre-dose baseline anti-CD3 antibody concentration by subtracting the mean baseline anti-CD3 antibody concentration from each post-dose value. It was determined by averaging the anti-CD3 antibody levels from three samples collected 30, 20 and 10 minutes before dosing. If the predose serum anti-CD3 antibody concentration was below the assay's quantitative level, it was not included in the average calculation. Pharmacokinetic parameters were determined from serum concentration data corrected for baseline anti-CD3 antibody concentration. Pharmacokinetic parameters were calculated by the model-independent method on a Digital Equipment Corporation VAX 8600 computer system using the latest version of BIOAVL software. The following pharmacokinetic parameters were determined: peak serum concentration (Cmax); time to reach peak serum concentration (tmax); area under the concentration-time curve (AUC) from time zero to the time of last blood collection calculated using the linear trapezoidal rule ( AUC0-72); and the terminal elimination half-life (t1/2) calculated from the elimination rate constant. Elimination rate constants were estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot. The mean, standard deviation (SD), and coefficient of variation (CV) of pharmacokinetic parameters for each treatment were calculated. The ratio of parameter means (preserved formulation/non-preserved formulation) was calculated.

Safety Results: The incidence of adverse events was equally distributed among treatment groups. There were no clinically significant changes from baseline or prestudy clinical laboratory tests or blood pressure, and there were no significant changes from prestudy in physical examination findings and vital sign measurements. The safety profiles should appear similar in both treatment groups.

Pharmacokinetic results: Mean serum anti-CD3 antibodies Fab-Folic Acid Bispecific for all 18 subjects at each measurement time point after receiving a single dose of one or more commercially available products specific for the same target antigen Concentration-time curves (not corrected for baseline anti-CD3 antibody levels) were compared with bispecific anti-CD3 antibodies containing non-naturally encoded amino acids. All subjects' predose baseline anti-CD3 antibody concentrations should be within the normal physiological range. Pharmacokinetic parameters were determined based on serum data corrected for mean baseline anti-CD3 antibody concentration prior to dosing, and Cmax and tmax were determined. The mean tmax of selected clinical comparators was significantly shorter than the tmax of bispecific anti-CD3 antibodies containing non-naturally encoded amino acids. The terminal half-life values of the commercially available anti-CD3 antibody products tested were significantly shorter compared to the terminal half-life of the bispecific anti-CD3 antibodies containing non-naturally encoded amino acids of the invention.

In summary, a single subcutaneously administered dose of a bispecific anti-CD3 antibody containing a non-naturally encoded amino acid will be safe and well tolerated by healthy female subjects. Based on the relative incidence of adverse events, clinical laboratory values, vital signs, and physical examination results, the safety profile of commercially available forms of anti-CD3 antibodies and bispecific anti-CD3 Fab folate antibodies containing non-naturally encoded amino acids will be equal to. Bispecific anti-CD3 antibodies containing non-naturally encoded amino acids may provide significant clinical utility to patients and healthcare providers.

It is to be understood that the embodiments and examples of the present invention are described for illustrative purposes only and that various modifications or changes thereto will occur to those skilled in the art and will be included within the spirit and scope of this application and the appended patent applications within the range. All publications, patents and patent applications cited herein are incorporated by reference in their entirety for all purposes.

<110> Hong Kong CHAMP PROFIT (CHINA) LIMITED

<120> Anti-CD3 antibody folic acid bioconjugate and its use

<140>TW 109106319

<141> 2020-02-26

<160> 62

<170> PatentIn version 3.5

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<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.1+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 32

<210> 33

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.4+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 33

<210> 34

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.5+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 34

<210> 35

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.6+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 35

<210> 36

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.1+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 36

<210> 37

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.2+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 37

<210> 38

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.3+CH1) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 38

<210> 39

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> LC (vL2.3+CL) is the light chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the light chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 39

<210> 40

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_regio

<223> HC (vH2.3+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 40

<210> 41

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.2+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 41

<210> 42

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH1.0+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 42

<210> 43

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.1+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 43

<210> 44

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.4+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 44

<210> 45

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.5+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 45

<210> 46

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.6+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 46

<210> 47

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.1+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 47

<210> 48

<211> 235

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.2+CH1- HK129pAF-DKTHT) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant , wherein unnatural amino acids (such as but not limited to pAF) are incorporated into this amino acid sequence

<400> 48

<210> 49

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.3+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 49

<210> 50

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.2+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 50

<210> 51

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH1.0+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 51

<210> 52

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.1+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 52

<210> 53

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.4+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 53

<210> 54

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.5+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 54

<210> 55

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH2.6+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 55

<210> 56

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.1+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 56

<210> 57

<211> 230

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> HC (vH3.2+CH1- HK129pAF) is the heavy chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the heavy chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant, where , there are unnatural amino acids (such as but not limited to pAF) incorporated into this amino acid sequence

<400> 57

<210> 58

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> LC (vL2.4+CL) is the light chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the light chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 58

<210> 59

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> LC (VL2.1+CL) is the light chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the light chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 59

<210> 60

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> LC (vL2.5+CL) is the light chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the light chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 60

<210> 61

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> LC (vL2.2+CL) is the light chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the light chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 61

<210> 62

<211> 217

<212> PRT

<213> Artificial sequence

<220>

<221> C_region+V_region

<223> LC (vL3.1+CL) is the light chain amino acid sequence of an anti-CD3 antibody, fragment or variant and/or the light chain amino acid sequence of an anti-CD3 Fab antibody, fragment or variant

<400> 62

N: Nitrogen atom

O:Oxygen atom

PEGx: x molecules of polyethylene glycol

Claims (23)

An anti-CD3 antibody or antibody fragment comprising: (a) any of the SEQ ID NOs: 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16 and 17 or; and (b) any of the SEQ ID NOs: 7, 8, 18, 19 and 20, wherein the anti-CD3 antibody or antibody fragment further comprises one or more folic acid and one or more PEG molecules. The anti-CD3 antibody or antibody fragment as described in claim 1, which includes any one of SEQ ID NO: 1-6 and any one of SEQ ID NO: 7-8. The anti-CD3 antibody or antibody fragment as described in claim 2, which includes any one of SEQ ID NO: 4-6 and any one of SEQ ID NO: 7-8. The anti-CD3 antibody or antibody fragment as described in claim 1, wherein the antibody or antibody fragment comprises IgG, Fv, Fab, (Fab')2 or single chain Fv (scFv). The anti-CD3 antibody or antibody fragment as described in any one of claims 1-4, wherein the antibody or antibody fragment comprises one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acids are parabens. Para-acetylphenylalanine, p -nitrophenylalanine, p -sulfotyrosine, p -carboxyphenylalanine, o-nitrophenylalanine o -nitrophenylalanine), m -nitrophenylalanine, p -boronylphenylalanine, o -boronylphenylalanine, m -boronylphenylalanine , p-aminophenylalanine ( p -aminophenylalanine), o-aminophenylalanine ( o -aminophenylalanine), meta-aminophenylalanine ( m -aminophenylalanine), p -acylphenylalanine, o-acylphenylalanine ( o -acylphenylalanine), m - acylphenylalanine, p -OMephenylalanine ( p -OMephenylalanine), o -OMe phenylalanine ( o -OMe phenylalanine), m -OMe phenylalanine, p Sulfophenylalanine ( p -sulfophenylalanine), o-sulfophenylalanine ( o -sulfophenylalanine), m -sulfophenylalanine, 5-nitrohistidine (5-nitro His), 3-nitro Tyrosine (3-nitro Tyr), 2-nitrotyrosine (2-nitro Tyr), nitro substituted Leu (nitro substituted Leu), nitro substituted His, Nitro substituted De (nitro substituted De), nitro substituted tryptophan (nitro substituted Trp), 2-nitro tryptophan (2-nitro Trp), 4-nitro Trp (4-nitro Trp), 5 -Nitro Trp (5-nitro Trp), 6-nitro Trp (6-nitro Trp), 7-nitro Trp (7-nitro Trp), 3-aminotyrosine (3-aminotyrosine), 2- Aminotyrosine (2-aminotyrosine), O-sulfotyrosine ( o -sulfotyrosine), 2-sulfooxyphenylalanine (2-sulfooxyphenylalanine), 3-sulfooxyphenylalanine, O-carboxyphenylalanine ( o -carboxyphenylalanine), m -carboxyphenylalanine), p-acetyl-L-phenylalanine ( p -acetyl-L-phenylalanine), p-propargyl-phenylalanine ( p -propargyl -phenylalanine), O-methyl-L-tyrosine ( o -methyl-L-tyrosine), L-3-(2-naphthyl)alanine) (L -3-(2-naphthyl)alanine), 3-methyl-phenylalanine, O -4-allyl-L-tyrosine , 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa (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 p-amino-L-phenylalanine), isopropyl-L-phenylalanine and p -propargyloxy-L-phenylalanine. The anti-CD3 antibody or antibody fragment of claim 5, wherein the non-naturally encoded amino acid is position-specifically incorporated into the antibody. The anti-CD3 antibody or antibody fragment of any one of claim 5, wherein the PEG is connected to the non-naturally encoded amino acid or folic acid. The anti-CD3 antibody or antibody fragment as described in any one of claims 1-4, wherein the PEG is covalently conjugated to a C-terminal heavy chain and/or light chain domain. The anti-CD3 antibody or antibody fragment of claim 8, wherein the PEG is conjugated to the C-terminal heavy chain and light chain domains via disulfide bond cross-linking. The anti-CD3 antibody or antibody fragment as described in claim 1, wherein the PEG is linear or branched. The anti-CD3 or antibody fragment of claim 8, wherein the heavy chain and light chain sequences comprise framework residues and/or germline mutations at one or more antigen binding positions. The anti-CD3 antibody or antibody fragment as described in claim 11, which is used for antigen binding optimization. The anti-CD3 antibody or antibody fragment as described in claim 1, further comprising a PEG linker molecular structure according to compounds 29A, 29B, 29C, 29D, 29E, 30A, 30B, 30C, 30D and 30E:

The anti-CD3 antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment contains one or more post-translational modifications. The anti-CD3 antibody or antibody fragment as described in any one of claims 1-4, which is used in therapy. The use of an anti-CD3 antibody or antibody fragment as described in any one of claims 1-4 in the manufacture of a medicament for optimized cell killing in cells expressing a high number of folate receptors, and one or Multiple non-naturally encoded amino acids are incorporated into the antibody. The use as described in claim 16, wherein the number of folate receptors is equal to or greater than 10,000. The use of an anti-CD3 antibody or antibody fragment as described in any one of claims 1-4 in the manufacture of a medicament for treating patients with cancer. The use of claim 18, wherein the cancer is characterized by high expression of folate receptor alpha (FOLR1), and wherein the cancer is an ovarian cancer. The use as claimed in claim 19, wherein the ovarian cancer includes an epithelial tumor, a stromal tumor, a germ cell tumor, a fallopian tube cancer or a primary peritoneal cancer. The use of the anti-CD3 antibody or antibody fragment as described in any one of claims 1-4 in the manufacture of a medicament for treating patients with diabetes. The use of the anti-CD3 antibody or antibody fragment as described in any one of claims 1 to 4 in the manufacture of a medicament for treating patients suffering from AIDS. A pharmaceutical Fab composition comprising a therapeutically effective amount of the anti-CD3 antibody or antibody fragment described in any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient.