TW202132336A - 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 folic acid bioconjugate and its use

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

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

Currently, there are deficiencies in the field of treatments targeting ovarian cancer and ovarian cancer types, including epithelial, mesenchymal and germ cell tumors and including fallopian tube cancer and primary peritoneal cancer. Although surgery, radiation and various chemotherapy are commonly used to treat patients with ovarian cancer, the U.S. Food and Drug Administration (FDA) has recently accepted bevacizumab (Avastin) as a first-line therapy for the treatment of advanced ovarian cancer supplementary biologics license Application (sBLA).

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

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

According to reports, ovarian cancer has an immunosuppressive environment, which makes the development of certain types of immunotherapies such as checkpoint inhibitors a challenge. There are currently no approved immunotherapies for the treatment of 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, kidney, 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, which is highly expressed in approximately 80-90% of ovarian cancers. FOLR1 combines folic acid (also known as vitamin B9) and 5-methyl-tetrahydrofolate (5-MTHF), the main metabolite of folic acid. High expression of FOLR1 is observed in approximately 76% of high-grade serous ovarian cancers (the main tissue type), compared to 11% in mucinous ovarian cancers. The expression of FOLR1 in normal tissues is low and restricted, which makes it a better target for tumor drug development.

In order to overcome the 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. The bispecific antibody may comprise an anti-CD3 antibody or consist of an anti-CD3 antibody, which has been engineered to have one or more on the heavy or light chain of the Fab (fragment antigen binding, antigen binding segment) Non-naturally encoded amino acid. The bispecific antibody or antibody fragment may comprise an anti-CD3 antibody that has been engineered to have one or more non-naturally encoded amino acids on the heavy chain and/or light chain.

The anti-CD3 bispecific antibody that recruits cytotoxic T cells to cancer cells is a promising new method for the treatment of various liquid and solid tumors. From one of the viewpoints, the present 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 chain or light chain of the Fab, preferably in the heavy chain. In one embodiment, the non-naturally encoded amino acid is para-acetyl phenylalanine (para-acetyl phenylalanine, where "phenylalanine" is abbreviated as F, so "para-acetyl phenylalanine" is abbreviated as Paf; in the present invention, And the English abbreviation of "Phenylalanine" is denoted by F, which is described 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 groups. The acid, optionally where two or more 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, the anti-CD3 Fab is optionally conjugated with both folic acid and polyethylene glycol (PEG) using a bifunctional linker. In one example, the anti-CD3 Fab is conjugated with two folic acid and two polyethylene glycol (PEG) molecules. In one embodiment, the conjugation is via the side chain of a non-naturally encoded amino acid such as pAF. In one example, anti-CD3 antibodies recruit cytotoxic T cells to folate receptor positive (FR+) tumor cells. In one example, the anti-CD3 antibody has improved efficacy, reduced toxicity, improved pharmacokinetic (PK) properties, improved affinity, improved tumor-associated antigen (TAA) binding, and improved half-life in vivo (T1/ 2) Improved in vitro activity, improved serum half-life and/or improved in vivo activity. In one example, the anti-CD3 antibody has improved efficacy. In one example, the anti-CD3 antibody has reduced toxicity. In one example, the anti-CD3 antibody has improved PK properties. In one example, the anti-CD3 antibody has improved affinity. In one example, the anti-CD3 antibody has improved TAA binding. In one example, the anti-CD3 antibody has improved T1/2 in vivo. In one example, the anti-CD3 antibody has improved in vitro activity. In one example, the anti-CD3 antibody has an improved serum half-life. In one example, 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) characteristics. 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. In order to achieve the best balance between efficacy and toxicity due to cytokine release syndrome (CRS), the inventors fine-tuned the affinity of anti-CD3 antibodies and optimized the binding of tumor-associated antigen (TAA). In order to increase the half-life (T1/2) in vivo, folic acid and PEG molecules of various sizes were simultaneously conjugated by using a bifunctional linker. The optimized conjugates show potent and selective in vitro activity, good serum half-life and potent in vivo activity in xenograft mouse models. This semi-synthetic method may be suitable for the use of small molecule ligands that are selective for other TAAs to generate additional anti-CD3 bispecific reagents.

From another point of view, 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 antibody of the present invention contains one or more non-naturally encoded amino acids. In one embodiment, the anti-CD3 antibody comprises a complete antibody heavy chain. In one example, the anti-CD3 antibody comprises a complete antibody light chain. In one embodiment, the anti-CD3 antibody comprises a complete antibody heavy chain and a complete antibody light chain. In one example, the anti-CD3 antibody comprises the variable region of the antibody light chain. In one embodiment, the anti-CD3 antibody comprises the variable region of the antibody heavy chain. In one example, the anti-CD3 antibody comprises the variable region of the light chain and the variable region of the 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 heavy chain of the anti-CD3 antibody. 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 example, the anti-CD3 antibody comprises three CDRs of the light chain. In one example, the anti-CD3 antibody comprises three CDRs of the heavy chain. In one example, 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 Fab. In one example, the anti-CD3 antibody contains two Fabs. In one embodiment, the anti-CD3 antibody comprises two or more Fabs. In one embodiment, the anti-CD3 antibody comprises scFv (single-chain variable fragment). In one example, the anti-CD3 antibody contains two scFvs. In one embodiment, the anti-CD3 antibody comprises two or more scFv. In one embodiment, the anti-CD3 antibody comprises a minibody. In one embodiment, the anti-CD3 antibody comprises two minibodies. In one embodiment, the anti-CD3 antibody comprises two or more minibodies. In one embodiment, the anti-CD3 antibody comprises a diabody. In one example, the anti-CD3 antibody comprises two diabodies. In one embodiment, the anti-CD3 antibody comprises two or more diabodies. In one embodiment, the anti-CD3 antibody comprises BiTE (Bi-specific T-cell engagers). In one example, the anti-CD3 antibody contains two BiTEs. In one embodiment, the anti-CD3 antibody comprises two or more BiTEs. In one embodiment, the anti-CD3 antibody comprises DART. In one embodiment, the anti-CD3 antibody contains two DARTs (Dual-affinity Re-targeting Antibody). In one embodiment, the anti-CD3 antibody comprises two or more DARTs. In one embodiment, the anti-CD3 antibody comprises TandAb (Tandem Diabody). In one example, the anti-CD3 antibody comprises two TandAbs. In one embodiment, the anti-CD3 antibody comprises two or more TandAbs. In one example, the anti-CD3 antibody comprises the variable region of the light chain and the variable region of the heavy chain. In one example, the anti-CD3 antibody contains a complete light chain and a complete heavy chain. In one embodiment, the anti-CD3 antibody comprises one or more Fc (Fragment crystallizable region) domains or parts thereof. In one embodiment, the anti-CD3 antibody comprises any combination of the above embodiments. In one embodiment, the anti-CD3 antibody comprises any of the homodimers, heterodimers, homomultimers or heteromultimers of the above-mentioned embodiments. In one embodiment, the anti-CD3 antibody comprises a polypeptide that binds to a binding partner, wherein the binding partner includes an antigen, a polypeptide, a nucleic acid molecule, a polymer, or other molecules or substances. In one example, 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 biologically active 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 example, the anti-CD3 antibody is linked to a polypeptide that is not an anti-CD3 antibody. In one example, an antigen-binding polypeptide containing a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides that may also contain a non-naturally encoded amino acid. In one example, an antigen-binding polypeptide containing a non-naturally encoded amino acid is linked to one or more polypeptide small molecule conjugates that may also contain a non-naturally encoded amino acid. In one example, an anti-CD3 antibody containing a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides that may also contain 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 comprises 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 contains two folic acid or two DUPA molecules. In one embodiment, the small molecule ligand comprises 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 the 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 the water-soluble polymer. In one embodiment, two small molecule ligands are connected to two water-soluble polymers. In an 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 folic acid 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 substitution in the anti-CD3 antibody may be a naturally-occurring or non-naturally-occurring amino acid, provided that at least one of the substitutions is a non-naturally-encoded amino acid.

In one embodiment, the non-naturally encoded amino acid contains a carbonyl group, an acetone group, an aminooxy group, a hydrazine group, a hydrazine group, a aminourea, an azide group, or an alkynyl group.

In one embodiment, the average molecular weight of the polyethylene glycol molecule is about 0.1 kDa to about 100 kDa. In one embodiment, the average molecular weight of the polyethylene glycol molecule is 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 can be about 5 kDa or about 10 kDa or about 20 kDa. For example, the average molecular weight of the polyethylene glycol polymer may be 5 kDa or 10 kDa or 20 kDa. In certain embodiments, the 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 2 kDa to 22 kDa, or 5 kDa to 20 kDa. 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 another point of view, the present invention also provides an anti-CD3 antibody polypeptide comprising a linker, polymer or biologically active molecule attached to one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acid The amino acid is incorporated into the polypeptide by the ribosome at a preselected position. Embodiments of the present invention provide anti-CD3 antibodies comprising at least one of SEQ ID NOs: 1-62. The anti-CD3 antibody includes both of SEQ ID NO: 1-62. Examples of the present invention provide anti-CD3 antibodies comprising both 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. Examples of the present invention provide anti-CD3 Fab antibodies comprising both of SEQ ID NO: 1-5. In other embodiments, the present 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 present 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, so 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 SEQ ID NO: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. In other embodiments, the present 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 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, 48, 49, 50, 50, 51, Any of 52, 53, 54, 55, 56, and 57. In other embodiments, the present 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 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, 48, 49, 50, 50, 51, Any one of 52, 53, 54, 55, 56 and 57; and SEQ ID NO: any one of 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, which comprises the amino acid sequence shown in one or more of SEQ ID NO: 1-62. In other embodiments, the present invention provides a cytotoxic active CD3 specific binding construct, which comprises the amino acid sequence shown in both of SEQ ID NOs: 1-62. In other embodiments, the present invention provides a cytotoxic active CD3 specific binding construct, which comprises 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, 46, 47, 48, 49, 50, 50, 51, 52, 53, 54, 55, 56 and 57. In other embodiments, the present invention provides a cytotoxic active CD3 Fab specific binding construct, which comprises an anti-CD3 Fab comprising SEQ ID NO: 7, 8, 9, 18, 19, 20, Any of 39, 58, 59, 60, 61, and 62. In other embodiments, the present invention provides a cytotoxic active CD3 specific binding construct, which comprises 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; and SEQ ID NO: 7, 8 , 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. Another embodiment of the present invention provides an anti-CD3 Fab antibody, wherein the anti-CD3 antibody comprises a binding domain comprising (a) a VH (heavy chain variable region) domain comprising a sequence selected from SEQ ID NO: 1-6 amino acid sequence, and (b) VL (light chain variable region) domain, which comprises an amino acid sequence selected from SEQ ID NO: 7-9.

From another viewpoint, another embodiment of the present invention provides an anti-CD3 antibody, wherein the antibody comprises one or more post-translational modifications. An embodiment of the present invention provides an anti-CD3 Fab antibody, wherein the antibody is linked to a linker, polymer, or biologically active molecule. Another embodiment of the present invention provides an anti-CD3 antibody, wherein the biologically active molecule is folic acid. Embodiments of the present invention provide anti-CD3 antibodies comprising one or more folic acid. Examples of the present invention provide anti-CD3 antibodies containing two folic acids. Another embodiment of the present invention provides an anti-CD3 antibody, wherein the biologically active molecule is DUPA. Embodiments of the present invention provide anti-CD3 antibodies comprising one or more DUPA. Examples of the present invention provide anti-CD3 antibodies comprising two DUPAs.

In certain embodiments, the anti-CD3 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, 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 one of 39, 58, 59, 60, 61, and 62.

In one embodiment, the anti-CD3 antibody comprises: 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, 48, 49 , 50, 50, 51, 52, 53, 54, 55, 56 and 57 of the amino acid sequence of any one of anti-CD3. 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 The anti-CD3.

In one embodiment, the anti-CD3 antibody comprises: 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, 48, 49 , 50, 50, 51, 52, 53, 54, 55, 56 and 57, and an anti-CD3 Fab containing the amino acid sequence of any one of SEQ ID NO: 7, 8, 9, 18, 19, 20, An anti-CD3 Fab of the amino acid sequence of any one of 39, 58, 59, 60, 61, and 62. Another embodiment of the present invention provides an anti-CD3 variant 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 NO: 7 and 10, or 7 and 14, or 7 and 11, or 7 and 15, or 7 and 12, or 7. And 16, or 7 and 13, or 7 and 17, or 7 and 1, or 7 and 18, or 7 and 19, or 12 and 18, or 12 and 19, or 12 and 16. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising SEQ ID NO: 7 and 10, or 7 and 14, or 7 and 11, or 7 and 15, or 7 and 12, or 7. And 16, or 7 and 13, or 7 and 17, or 7 and 1, or 7 and 18, or 7 and 19, or 12 and 18, or 12 and 19, or 12 and 16, each of which contains at least one unnatural Amino acid. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab comprising SEQ ID NO: 7 and 10, or 7 and 14, or 7 and 11, or 7 and 15, or 7 and 12, or 7. And 16, or 7 and 13, or 7 and 17, or 7 and 1, or 7 and 18, or 7 and 19, or 12 and 18, or 12 and 19, or 12 and 16, each of which contains two unnatural Amino acid. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab, the anti-CD3 Fab comprises 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 includes at least one unnatural amino acid. In one embodiment, the anti-CD3 antibody comprises an anti-CD3 Fab, and the anti-CD3 Fab comprises SEQ ID

NO: 12, which includes at least one unnatural amino acid; and one of SEQ ID NO: 18, or 19, or 16, which includes at least one unnatural 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, and the antibody comprises SEQ ID NO: 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 may 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 present invention provide anti-CD3 Fab antibodies selected from SEQ. ID. NO: 1 to 62, in which non-naturally encoded amino acids are incorporated. In one example, the non-naturally encoded amino acid is site-specifically incorporated into the antibody.

In other embodiments, the present invention provides an anti-CD3 Fab antibody, wherein the non-naturally encoded amino acid is selected from: 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 ( o -4-allyl-L-tyrosine), 4-propyl-L-tyrosine (4-propyl-L-tyrosine), p-propargyloxy-L-phenylalanine, tri-O-acetyl GlcNAc-serine, L-dopa (L-DOPA), fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine (p -azido-L-phenylalanine) , acyl -L- phenylalanine (p-acylphenylalanine), benzoyl group -L- phenylalanine (p -benzoyl-L-phenylalanine) , L- serine phosphorylation pairs (L-phosphoserine), phosphonoserine, phosphonotyrosine, p- iodo-phenylalanine, p- bromophenylalanine, p-amino -L -phenylalanine (p -amino-L-phenylalanine) or isopropyl-L-phenylalanine.

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

From another viewpoint, another embodiment of the present invention provides an anti-CD3 Fab variant in which the heavy chain further includes an amino acid extension at the C-terminus. An embodiment of the present invention provides an anti-CD3 Fab variant, wherein the amino acid extension comprises the amino acid DKTHT. Another embodiment of the present invention provides an anti-CD3 Fab variant, which further comprises an amino acid extension at the C-terminus of the heavy chain. Other embodiments of the present invention provide an anti-CD3 Fab variant in which the amino acid extension comprises the amino acid DKTHT.

Another embodiment of the present invention provides an anti-CD3 Fab antibody, 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 contains mouse framework residues at positions 30, 49, 77 or 93 (according to Kabat numbering). Another embodiment of the present invention provides an anti-CD3 Fab antibody, 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 positions 36, 46, 49, 57 or 58 (according to Kabat numbering).

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

From another point of view, in another embodiment, the present invention provides a method for optimizing cell killing in cells expressing high folate receptor numbers, the method comprising an anti-CD3 Fab antibody, wherein the antibody comprises one or Multiple folates; and one or more non-naturally encoded amino acids are incorporated into the antibody. In another embodiment, the present invention provides a method for optimizing the killing of tumor cells expressing high folate receptor numbers by redirected T cells, the method comprising an anti-CD3 Fab antibody, wherein the antibody comprises one or more Folic acid; and 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 present invention provides a method, wherein the water-soluble polymer comprises polyethylene glycol. In another embodiment, a method is provided in which the water-soluble polymer is linear or branched. Polyethylene glycol is 1 kDa to 100 kDa. 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 another point of view, another embodiment of the present invention provides a method of reducing cytotoxicity in cells. Another embodiment of the present invention provides a method for reducing cytotoxicity in cells, which is achieved by optimizing or reducing the affinity of anti-CD3 antibodies to their targets for T cell recruitment. Another embodiment of the present invention provides a method for increasing the cytotoxicity of tumor cells by conjugating more than one folic acid before T cell binding to preferentially bind to tumor cells. Another embodiment of the present invention provides a method for improving the serum half-life of an anti-CD3 Fab antibody. Another embodiment of the present invention provides a method for improving the serum half-life of anti-CD3 Fab antibodies by conjugated Fab with pharmacokinetic (PK) extension molecules including but not limited to HSA, C12-C16 acyl chain, XTEN or water-soluble polymer 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 characteristics, improving affinity, improving TAA binding, improving T1/2 in vivo, improving in vitro activity, improving serum half-life and/or improving in vivo activity. Another embodiment provides a method of improving efficacy. Another embodiment provides a method of reducing toxicity. Another embodiment provides a method for improving PK characteristics. Another embodiment provides a method for improving affinity. Another embodiment provides a method of improving TAA binding. Another embodiment provides a method for improving T1/2 in vivo. Another embodiment provides a method for improving in vitro activity. Another embodiment provides a method for improving in vivo activity.

From another viewpoint, another embodiment of the present invention provides an anti-CD3 Fab antibody, wherein the heavy chain or light chain sequence contains human germline mutations at one or more positions. The embodiment of the present invention provides an anti-CD3 Fab antibody, wherein the human germline mutation in the heavy chain sequence is at position 35 or 52 (according to Kabat numbering). Another embodiment of the present invention provides an anti-CD3 Fab antibody, wherein the human germline mutation in the light chain sequence is at position 53 (according to Kabat numbering).

Another embodiment of the present 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 another point of view, in another embodiment, the present invention provides a method of treating a patient having a disease or disorder in cells expressing high folate receptor numbers, the method comprising administering to the patient a therapeutically effective amount The disclosed anti-CD3 Fab antibody of the present invention. In another embodiment of the present invention, a bispecific anti-CD3 Fab comprising two folic acid and two PEGylated molecules is provided. In another embodiment, the bispecific anti-CD3 Fab antibody further comprises a non-naturally encoded amino acid site specifically incorporated. The present invention provides a method of treating cancer, which is achieved by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. In one embodiment, the cancer is ovarian cancer. In one embodiment, the ovarian cancer is epithelial, mesenchymal and germ cell tumors. 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, which is achieved by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. The present invention provides a method for treating AIDs, which is achieved by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. The present invention provides a method for treating diabetes, which is achieved by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. The anti-CD3 antibody of the present invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is conjugated with two folic acid and two PEGylated molecules. In another embodiment, the non-naturally encoded amino acid is position-specifically incorporated into the anti-CD3 Fab antibody, and is combined with two folic acid and two polyethylene glycol via the side chain of the non-natural amino acid. Conjugation of alcoholized molecules.

The anti-CD3 antibodies of the present invention are used to treat diseases or disorders in cells expressing a high number of folate receptors. The anti-CD3 antibody of the present invention is used to treat cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, ovarian cancer is epithelial, mesenchymal, and germ cell tumors. 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 antibody of the present invention is used to treat genetic diseases. The anti-CD3 antibody of the present invention is used to treat AIDS. The anti-CD3 antibody of the present invention is used to treat diabetes. The anti-CD3 antibody of the present invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is conjugated with two folic acid and two PEGylated molecules. In another embodiment, the non-naturally encoded amino acid is position-specifically incorporated into the anti-CD3 Fab antibody, and the conjugation with two folic acid and two PEGylated molecules is via the non-natural amino acid. The acid side chain is realized. The anti-CD3 antibody of the present invention can be used to manufacture drugs for treating diseases or disorders in cells expressing a high number of folate receptors. The anti-CD3 antibody of the present invention can be used to manufacture drugs for the treatment of cancer. In one embodiment, the cancer is ovarian cancer. In one embodiment, ovarian cancer is epithelial, mesenchymal, and germ cell tumors. 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 antibody of the present invention can be used to manufacture drugs for the treatment of genetic diseases. The anti-CD3 antibody of the present invention can be used to manufacture drugs for the treatment of AIDS. The anti-CD3 antibody of the present invention can be used to manufacture drugs for the treatment of diabetes. The anti-CD3 antibody of the present invention may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody is conjugated with two folic acid and two PEGylated molecules. In another embodiment, the non-naturally encoded amino acid is position-specifically incorporated into the anti-CD3 Fab antibody, and the conjugation with two folic acid and two PEGylated molecules is via the non-natural amino acid. The acid side chain is realized.

The present disclosure provides an anti-CD3 antibody or antibody fragment or variant, which has 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, trisomy, tetrasomy, bifunctional hetero Combine 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, 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. The present invention discloses herein a bispecific antibody conjugate comprising an anti-CD3 Fab antibody or antibody fragment, in which one or more non-naturally encoded amino acids are incorporated. The present invention further discloses a bispecific antibody conjugate comprising an anti-CD3 Fab antibody or antibody fragment and one or more folate molecules, wherein one or more non-naturally encoded amino acids are specifically incorporated into anti-CD3 Fab antibody. The present invention discloses a bispecific antibody conjugate comprising an anti-CD3 Fab antibody or antibody fragment, one or more folate molecules and one or more PEG molecules, wherein one or more non-naturally encoded amino acids are position-specific Sexually incorporated into the anti-CD3 Fab antibody. The present invention discloses an anti-CD3 Fab bispecific antibody, which comprises: an antibody or antibody fragment; one or more small molecules; and one or more linkers, wherein the antibody or antibody fragment is connected to The one or more small molecules are connected, and wherein the small molecules are one or more folic acid or one or more DUPA molecules or analogs or derivatives. The antibody or antibody fragment can be position-specifically linked to one or more folic acid or one or more DUPA molecules via one or more linkers. The antibody or antibody fragment may contain one or more unnatural amino acids. The antibody or antibody fragment can be linked to one or more folic acid or one or more DUPA molecules via one or more linkers that are linked to one or more unnatural amino acids. Non-natural amino acids can be incorporated into antibodies site-specifically. The antibody or antibody fragment can be linked to one or more folate or DUPA molecules via one or more PEG molecules that are linked to one or more non-natural 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 that are linked to natural amino acids. The antibody or antibody fragment may be an anti-CD3 Fab.

The present invention discloses a bispecific antibody conjugate, which comprises: an anti-CD3 Fab; one or more folate molecules; and one or more linkers, wherein the anti-CD3 Fab is connected to the one or more linkers through the one or more linkers. One or more folate molecules are linked. The anti-CD3 Fab may contain one or more non-natural amino acids. The one or more non-natural amino acids can replace the natural amino acids of the anti-CD3 Fab. The present invention discloses a bispecific antibody conjugate, which comprises: anti-CD3 Fab; one or more DUPA molecules; and one or more linkers, wherein the anti-CD3 Fab is connected to the one or more linkers through the one or more linkers. One or more DUPA molecules are linked. The anti-CD3 Fab may contain one or more unnatural amino acids. The one or more non-natural amino acids can replace the natural amino acids of the anti-CD3 Fab.

The novel features of the present invention are specifically described in the scope of the attached patent application. A better understanding of the features and advantages of the disclosure of the present invention will be obtained by referring to the following detailed description and the accompanying drawings. The following detailed description sets forth exemplary embodiments that utilize the principles of the present invention.

It should be understood that the present invention is not limited to the specific methods, laboratory protocols, cell lines, constructs, and reagents described in the present invention, and can therefore vary. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is only limited by the scope of the attached patent application.

definition:

As used in the scope of the present invention and the appended application, a reference without a specific number includes a plurality of 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 their equivalents known to those skilled in the art, and the like. The terms "PEG-folate" or "folate-PEG" and the terms "bisPEG-difolate" or "difolate-bisPEG" are used interchangeably in the present invention.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods, devices and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, the present invention currently only describes preferred methods, devices and materials.

All publications and patents mentioned in the present invention are incorporated by reference into the present invention to describe and disclose, for example, the structures and methods described in the publications, which can be used in combination with the presently described invention. The publications discussed in this invention are provided only with regard to their disclosures prior to the filing date of this application. Nothing in the present invention should be construed as an admission that the inventor has no right to precede the disclosure due to prior inventions or for any other reason.

The term "substantially purified" refers to an anti-CD3 Fab antibody, which may be substantially or essentially free of the groups normally associated with or interacting with the protein found in its naturally occurring environment. The naturally occurring environment is a natural cell or a host cell in the case of a recombinantly produced anti-CD3 antibody. Anti-CD3 antibodies that may be substantially free of cellular material include protein preparations having 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 the anti-CD3 antibody or its variant is recombinantly produced by the host cell, the protein may be about 30%, about 25%, about 20%, about 15%, about 10% of the dry weight of the cells. It is present in an amount of about 5%, about 4%, about 3%, about 2%, or about 1% or less. When the anti-CD3 antibody or its variant (variant) is recombinantly produced by the host cell, the protein may be about 5g/L, about 4g/L, about 3g/L, about 2g of the 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 exist in the periplasm ( periplasm) and/or medium. Therefore, a "substantially purified" anti-CD3 antibody as produced by the method of the present invention may 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% , At least about 60%, at least about 65%, at least about 70% purity level, in particular, at least about 75%, 80%, 85% purity level, and more particularly, at least about 90% purity level, at least A purity level of about 95%, a purity level of at least about 99% or higher, 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 exogenous polynucleotides, 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 it can be integrated into the host genome.

Antibodies are proteins that exhibit binding specificity for specific antigens. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bond connections varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain 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 constant domains at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable structure of the light chain The domain is aligned with the variable domain 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 parts of variable domains vary greatly in sequence between antibodies and are responsible for the binding specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called Complementarity Determining Regions (CDR) in the variable domains of both the light chain and the heavy chain. The highly conserved parts of variable domains are called framework regions (FR). The variable domains of the natural heavy chain and light chain each contain four FR regions, mainly adopting a β-sheet configuration, connected by three CDRs, which form a loop connecting the β-sheet structure, And in some cases it forms 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 formation of the antigen binding site of the antibody (see Kabat et al., "Protein Sequences of Immunological Concern" "(Sequences of Proteins of Immunological Interest), 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions. According to the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be divided into different categories. 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 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 IgG1, 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, the affinity maturation of antibodies is driven by antigen selection of higher affinity antibody variants, which are mainly produced by somatic supermutation. A "repertoire shift" also often occurs, in which the main germline genes of the secondary or tertiary response appear to be different from the primary or secondary response.

By introducing mutations into antibody genes in vitro and using affinity selection to isolate mutants with improved affinity, the affinity maturation process of the immune system can be replicated. Such mutant antibodies can be displayed on the surface of filamentous phage or microorganisms such as Escherichia coli and yeast, and antibodies can be selected by their affinity for the antigen or by the kinetics of their dissociation from the antigen (dissociation rate). Hawkins et al., J. Mol. Biol. 226:889-896 (1992). CDR walking mutagenesis has been used to mature the affinity of human antibodies that bind to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) (Barbas III et al., PNAS (USA) 91 : 3809-3813 (1994); 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)). Using antibody chain shuffling and CDR mutagenesis to mature the affinity of high-affinity human antibodies against the third hypervariable loop of HIV (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 variable region genes are simultaneously and thoroughly searched for improved mutations. body. Use the initial limited mutagenesis strategy to make the αvβ3-specific (αvβ3-specific) humanized antibody affinity maturation, in which each position of all six CDRs is mutated, and then expression and screening contain the highest affinity Combinatorial library of mutants (Wu et al., PNAS (USA) 95: 6037-6-42 (1998)). Phage display antibodies are reviewed in the following documents: Chiswell and McCafferty TIBTECH 10:80-84 (1992); and Rader and Barbas III Current Opinion in Biotech. 8:503-508 (1997). In the above reference, a mutant antibody with improved affinity compared to a parent antibody in each case is reported, and the mutant antibody has an amino acid substitution in the CDR.

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

The "antibody" in the present invention refers to a protein consisting of one or more polypeptides substantially encoded by all or part of an antibody gene. 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 countless immunoglobulin variable region genes. The antibodies in the present invention are meant to include full-length antibodies and antibody fragments, and include antibodies that are naturally occurring or engineered in any organism (e.g., variants). The antibodies disclosed in the present invention may be human antibodies, humanized antibodies, engineered antibodies, non-human antibodies and/or chimeric antibodies. Humanized antibodies and methods for their preparation are well known in the art. (See, for example, U.S. Patent Nos. 5,821,337; 7,527,791; 6,982,321; 7,087,409; 5,766,886). Generally, a humanized antibody contains one or more variable domains, where the CDR or part thereof is derived from a non-human antibody, and the framework region or part thereof is derived from a human antibody sequence. The humanized antibody may optionally comprise at least a part of a human constant region. In one embodiment, for example, framework residues in a humanized antibody can be replaced with corresponding residues from a non-human antibody to restore or improve antibody specificity or affinity. Human antibodies are antibodies from which CDR residues are derived. Chimeric antibodies may refer to antibodies produced by combining or joining two or more antibody genes that originally coded for 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 an embodiment, at least a part of the antibody or antibody fragment may be from human or cynomolgus monkey (cynomolgus monkey), but is not limited thereto. In certain embodiments, the antibodies disclosed in the present invention may have cross-species reactivity, for example, the antibodies can recognize human antigens and cynomolgus monkey antigens (for example, a human/cyno antibody (a human/cyno antibody)).

"Antibody fragment" refers to any form of antibody other than the full-length form. The antibody fragments of the present invention include antibodies of smaller components 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), diabodies, tribodies, tetrabodies, 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. (Maynard and Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Unless specifically stated otherwise, statements and patent claims using the term "antibody" may specifically include "antibody fragments." In certain embodiments, "anti-CD3 antibody", "anti-CD3 Fab", "anti-CD3 Fab antibody" and "anti-CD3 Fab variant" are antibody fragments as defined in the present invention.

The "computational screening method" in the present invention refers to any method used to design one or more mutations in a protein, wherein the method uses a computer to evaluate potential amino acid side chain substitutions between 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 antibody H and/or L chain. In most mammals, including humans and mice, this form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having a light chain and a heavy chain, and each light chain The chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, Cγ1, Cγ2, and Cγ3. In each pair, the light chain 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 antibody effector functions. In certain mammals, such as camels and llamas, full-length antibodies may consist of only two heavy chains, each of which contains the immunoglobulin domains VH, Cγ2, and Cγ3.

The "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 composed 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 in the present invention, "variant protein sequence" refers to a protein sequence having one or more residues that differ from another similar protein sequence in amino acid identity. The similar protein sequence may be a natural wild-type protein sequence, or another variant of the wild-type sequence. Generally, the starting sequence is referred to as the "parent" sequence, and can be a wild-type or variant sequence. For example, preferred embodiments of the present invention can utilize humanized parent sequences on which computational analysis is performed to generate variants.

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

Regarding the anti-CD3 Fab antibody of the present invention, the term "antigenically specific" or "pecifically binds" refers to one or more expressions with an antigen of interest or a binding partner (binding partner). Epitopes are anti-CD3 antibodies that bind but do not substantially recognize and bind other molecules in a sample containing a mixed population of antigens.

As used in the present invention, the term "bispecific anti-CD3 antibody" or "multispecific anti-CD3 antibody" refers to an anti-CD3 antibody comprising two or more antigen binding sites or binding partner binding sites, the first binding site It has affinity for a first antigen or epitope (epitopes) and the second binding site has binding affinity for a second antigen or epitope (epitopes) that is different from the first antigen or epitope. In one example, the bispecific anti-CD3 antibody has a binding site that binds to CD3 and one or more binding sites that have binding affinity for different antigens or epitopes.

As used in the present invention, the term "epitope" refers to a position on an antigen or binding partner that is recognized by an anti-CD3 Fab antibody. If the antigen contains a polypeptide, the epitope may be a linear or conformational amino acid sequence or shape. The epitope can also be any position on any type of antigen that enables the anti-CD3 antibody to bind to the antigen.

As used in the present invention, "antigen-binding polypeptide" or "anti-CD3 Fab antibody" shall include at least those polypeptides and proteins that have the biological activity of specific binding to a specific binding partner. Such as antigens, as well as CD3 analogs, CD3 isoforms, CD3 mimetics, CD3 fragments, hybrid CD3 proteins, their fusion proteins, oligomers and polymers, homologs (homologues), glycosylation pattern variants and mutant proteins, regardless of their biological activity, in addition to their synthesis or manufacturing methods, including but not limited to recombination (whether from cDNA, genomic DNA, synthetic DNA or other forms Nucleic acid production), in vitro, in vivo, through microinjection of nucleic acid molecules, synthesis, transgene 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, Fabs, scFvs, 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 (prodrugs), as well as prodrugs, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants and naturally-occurring human antibodies of the salts. Stereoisomers of CD3 antibodies and agonists, mimetics and antagonist variants of naturally occurring human anti-CD3 antibodies and their polypeptide fusions. The term "antigen-binding polypeptide" encompasses fusions containing additional amino acids at the amino terminus, carboxy terminus, or both. Exemplary fusions include, but are not limited to, for example, methionyl anti-CD3 antibody (methionyl anti-CD3 antibody), in which methionine is linked to the N-terminus of an anti-CD3 antibody produced by recombinant expression, a fusion for purification purposes (Including but not limited to polyhistidine or affinity epitopes), fusions for the purpose of linking anti-CD3 antibodies with other biologically active molecules, fusions with serum albumin binding peptides, and with serum proteins such as Fusion of serum albumin.

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

The naturally produced antibody (Ab) is a tetrameric structure 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 a variable domain (VL) and a constant domain (CL), and each heavy chain has a variable domain (VH) and three constant domains (CH). Each domain composed of about 110 amino acid residues is folded into a characteristic-sandwich structure formed by two β-sheets stacked on each other, that is, an immunoglobulin fold. The VL domains each have three complementarity determining regions (CDR1-3), and the VH domains each have at most three complementarity determining regions (CDR1-3), which are loops or loops connected to the β strand at one end of the domain. The variable regions of both the light chain and the heavy chain 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 a large number of molecules.

The functional substructures of Ab can be prepared by proteolysis and recombinant methods. They include Fab fragments, Fv fragments, and Fc parts. The Fab fragments contain the VH-CH1 domain of the heavy chain and the VL-CL1 domain of the light chain joined by a single interchain disulfide bond, while the Fv fragment only contains VH and VL domains, the Fc portion contains the non-antigen binding region of the molecule. In some cases, a single VH domain retains a 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). Sometimes it is found that the isolated light or heavy chain also retains 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 immunoglobulin heavy and light chains covalently linked by a peptide linker (Sz Hu et al., Cancer Research, 56, 3055-3061, 1996). These small (Mr 25,000 Da) proteins usually retain specificity and affinity for antigens in a single polypeptide, and can provide suitable structural units for larger antigen-specific molecules. In many cases, the short half-life of scFv in the loop limits their therapeutic utility.

Using a part of the Ig VH domain as a template, a small protein scaffold called "minibody" was designed (Pessi et al., Nature 362, 367-369, 1993). By randomizing the loops corresponding to CDR1 and CDR2 of VH, and then using the phage display method (phage display method) to select mutants to identify the high affinity for interleukin-6 (dissociation constant (Kd) is about 10-7 M) (Martin et al., EMBO J. 13, 5303-5309, 1994).

When analyzing IgG-like substances in camel serum, camels usually lack variable light chain domains, which indicates that sufficient antibody specificity and affinity can be generated from only VH domains (three or four CDR loops). A "camelized" VH domain with high affinity has been prepared, and high specificity can be generated by randomizing only CDR3.

The alternative to "mini antibody" is "diabody". Diabodies are small bivalent and bispecific antibody fragments with two antigen binding sites. The fragment comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). The size of the diabody is similar to that of the Fab fragment. By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create 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 mimics have been prepared (Dougall et al., 1994, Trends Biotechnol. 12, 372-379). CDR peptides are short, usually cyclic peptides, which correspond to the amino acid sequence of the CDR loop of an antibody. The CDR loop is responsible for the antibody-antigen interaction. 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 transplanted to the human Ig framework without loss of affinity (Jones et al., Nature 321, 522-525, 1986; Riechmann et al., 1988).

In humans, a specific Ab is selected from a large library and amplified (affinity maturation). These processes can be reproduced in vitro using combinatorial library technology. The successful display of Ab fragments on the surface of phage makes 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). Through this technology, more and more Fab and Fv (and their derivatives) have been produced. Combination technology can be combined with Ab analogs (mimics).

Many protein domains that may be used as protein scaffolds have been expressed as fusions with phage capsid proteins. It is reviewed in Clackson and Wells, Trends Biotechnol. 12:173-184, 1994. Some of these protein domains have been used as scaffolds to display 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 1:70-75 1994) and streptococcal IgG binding domain (O'Neil et al., Techniques in Protein Chemistry V (Crabb, L ed.)), pages 517-524, Academic Press, San Diego, 1994). These scaffolds show a single randomized ring or area. Amylase aprotin has been used as a presentation scaffold on the filamentous bacteriophage M13 (McConnell and Hoess, J. Mol. Biol. 250:460-470, 1995).

The covalent attachment of hydrophilic polymer polyethylene glycol (abbreviated as PEG) is for many biologically active molecules (including proteins, peptides, especially hydrophobic molecules) to increase water solubility, bioavailability, and prolong serum half-life. Methods of treatment half-life, regulation of immunogenicity, regulation of biological activity, or extension of cycle time. PEG has been widely used in drugs, artificial implants, and other applications where biocompatibility, non-toxicity, and non-immunogenicity are important. In order to maximize the desired properties of PEG, the total molecular weight and state of hydration of the one or more PEG polymers attached to the biologically active molecule must be high enough to impart advantageous properties normally associated with PEG polymer attachment, such as increased water solubility And circulatory half-life without adversely affecting the biological activity of the parent molecule.

PEG derivatives are usually linked to biologically active molecules through reactive chemical functional groups (such as lysine, cysteine and histidine residues, N-terminus and carbohydrate moieties). Proteins and other molecules often have a limited number of reaction sites available for polymer attachment. Generally, the most suitable position for modification via polymer attachment plays an important role in receptor binding and is necessary to maintain the biological activity of the molecule. As a result, the 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 a conjugate with sufficient polymer molecular weight to give the target molecule the desired advantages, prior art methods usually involve randomly attaching many polymer arms to the molecule, thereby increasing the reduction or even completeness of the biological activity of the parent molecule. The risk of loss.

The reaction position that forms the position where the PEG derivative is attached to the protein is determined by the structure of the protein. Proteins (including enzymes) are composed of various α-amino acid sequences, and the general structure of the α-amino acid is H2N--CHR--COOH. The α-amino part (H2N--) of an amino acid joins with 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 equal to hundreds or thousands. The fragment represented by R may contain reaction sites for protein biological activity and for attachment of PEG derivatives.

For example, in the case where the amino acid is a lysine acid, there is a —NH2 moiety in the ε position and the α position. ε--NH2 can react freely under alkaline pH conditions. Many existing technologies in the field of protein derivatization with PEG are devoted to the development of PEG derivatives to attach them to the epsilon-NH2 moiety of lysine residues present in proteins. ("Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, pages 1-17, 2003). However, these PEG derivatives all have a common limitation, that is, they cannot be selectively installed on the numerous lysine residues present on the protein surface. In the case where lysine residues are important for protein activity (e.g., they are present in the active position of an enzyme) or lysine residues play a role in mediating the interaction of the protein with other biological molecules (as in The same is true for the receptor binding position), which may be a major limitation.

The second and equally important complication of existing methods for protein PEGylation is that PEG derivatives may have undesirable side reactions with undesirable residues. Histidine contains a reactive imine moiety, represented in the structure as --N(H)--, but many chemically reactive substances that react with ε--NH2 can also react with --N(H)-- reaction. Similarly, the side chain of the amino acid cysteine has a free sulfhydryl group, which is structurally represented as -SH. In some cases, PEG derivatives directed to 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 molecules. It is desirable to develop PEG derivatives that allow the introduction of chemical functional groups at a single location within a protein, which can thus enable one or more PEG polymers to be selectively conjugated to biologically active molecules at specific and predictable locations on the protein surface.

In addition to lysine residues, considerable efforts in the art have also been devoted to the development of activating PEG reagents that target other amino acid side chains (including cysteine, histidine, and N-terminus). See, for example, U.S. Patent No. 6,610,281, which is incorporated herein by reference, and "Polyethylene Glycol and Derivatives for Advanced PEGylation", Nektar Molecular Engineering Catalog, 1-17 Page, 2003. Site-directed mutagenesis and other techniques known in the art can be used to introduce cysteine residues positionally into the protein structure, and the resulting free sulfhydryl moiety can be reacted with thiol-reactive functional groups. The PEG derivative reacts. However, this method is complicated because the introduction of free sulfhydryl groups will complicate the expression, folding and stability of the resulting protein. Therefore, it is desirable to have a method of introducing chemical functional groups into biologically active molecules, which can selectively conjugate one or more PEG polymers to proteins, while being compatible with sulfhydryl groups and other chemical functional groups commonly found in proteins. (Ie not participating in undesirable side reactions).

As pointed out in the art, many such derivatives have been developed for attachment to the side chain of proteins, especially on the lysine amino acid side chain -NH2 moiety and cysteine side chain The -SH part of the said derivatives has proved to be problematic in the synthesis and use of the derivatives. Some derivatives form unstable bonds with proteins, and these bonds are easily hydrolyzed and therefore decompose, degrade, or are otherwise unstable in an aqueous environment (for example, in blood). Some derivatives form more stable bonds, but undergo hydrolysis before forming the bonds, which means that the reactive groups on the PEG derivatives may be inactivated before the protein can be attached. Some derivatives are slightly toxic, so they are not suitable for use in the body. Some derivatives react too slowly to be practical. Some derivatives cause loss of protein activity by attaching to positions responsible for protein activity. Some derivatives are not specific at the position where they will be attached, which may also lead to loss of desired activity and lack of reproducibility of results. In order to overcome the challenges associated with partially modifying proteins with polyethylene glycol, more stable PEG derivatives have been developed (e.g., U.S. Patent 6,602,498, which is incorporated into the present invention by reference) or selected with thiol moieties on molecules and surfaces. Sexually reactive PEG derivatives (e.g., U.S. Patent 6,610,281, which is incorporated herein by reference). It is obvious in the art that there is a need for PEG derivatives that are chemically inert in a physiological environment until required to react selectively to form stable chemical bonds.

Recently, a new technology in protein science has been reported, which is expected to overcome many limitations related to the position-specific modification of proteins. Specifically, new components have been added to the prokaryotic Escherichia coli ( Escherichia coli or E. coli ) (for example, L. Wang et al., Science 292:498-500, 2001) and the eukaryotic Saccharomyces cerevisiae ( Saccharomyces cerevisiae). Or S. cerevisiae ) (for example, J. Chin et al., Science 301:964-7, 2003) in the protein biosynthesis mechanism, which enables the incorporation of non-gene-encoded amino acids into proteins in the body. Many new amino acids with novel chemical, physical or biological properties, including photoaffinity tags and photoisomerizable amino acids, keto amino acids and glycosylated amino acids, have been used in response to this method The amber codon TAG is effectively and with high fidelity incorporated 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 have shown that chemical functional groups such as ketone, alkynyl and azide can be selectively and routinely introduced, which are not present in proteins and are chemically inert to all functional groups present in 20 common genetically encoded amino acids , And can be used to efficiently and selectively react 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 Acid imine groups provide valuable alternatives. It is known that certain chemical functional groups are inert to the functional groups present in the 20 common genetically encoded amino acids, but they can react cleanly and efficiently to form stable bonds. For example, it is known in the art that azide and acetylene groups undergo the Huisgen [3+2] cycloaddition reaction in the presence of a catalytic amount 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 functional groups that are chemically inert to the amine, sulfhydryl, carboxylic acid, and hydroxyl groups present in the protein but can also react smoothly and effectively with the acetylene moiety to form a cycloaddition product. It is important that in the absence of the acetylene moiety, the azide remains chemically inert and does not react 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 the anti-CD3 antibody as described above, and a polypeptide that retains at least one biological activity of a naturally-occurring antibody (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 with polymers such as PEG, and may include cysteine, lysine, N-terminal or C-terminal amino acids or other residues One or more additional derivatization. In addition, 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 use known in the art The technology is conjugated with naturally-encoded amino acids, such as lysine or cysteine. US Patent No. 4,904,584 discloses a pegylated lysine acid-depleted polypeptide 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 with 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 with the protein. WO 99/03887 discloses PEGylated variants of polypeptides belonging to the growth hormone superfamily, in which cysteine residues have been substituted with non-essential amino acid residues located in designated regions of the polypeptide. WO 00/26354 discloses a method for producing glycosylated polypeptide variants with reduced allergenicity, which variants comprise at least one additional glycosylation site 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, and N-linked or O-linked glycosylated forms of the polypeptide. Variants containing single nucleotide changes are also considered to be biologically active variants of anti-CD3 antibodies. In addition, splice variants are also included. The term "antigen-binding polypeptide" also includes any one or more anti-CD3 antibodies or any other polypeptides, proteins, carbohydrates, polymers, small molecules, linkers, ligands, or any type of other biologically active molecules of anti-CD3 antibodies. Polymers, homodimers, heteromultimers or homomultimers (connected by chemical means or expressed as fusion proteins), and polypeptide analogs that contain, for example, specific deletions or other modifications but still 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, addition, substitution or deletion can adjust one or more characteristics or activities of the anti-CD3 antibody, including but not limited to adjusting the affinity to the antigen, adjusting (including but not limited to increasing or decreasing) the antigen configuration or other secondary and tertiary levels. Or quaternary structural changes, stabilize antigen configuration or other secondary, tertiary or quaternary structural changes, induce or cause antigen configuration or other secondary, tertiary or quaternary structural changes, regulate circulatory half-life, and regulate therapeutic half-life , Adjust the stability of the polypeptide, adjust the dosage, adjust the release or bioavailability, promote purification, or improve or change the specific route of administration. Similarly, the antigen-binding polypeptide may include 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 characteristics 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 side The chain is directly connected to the same or different non-naturally encoded amino acid side chain, directly connected to the naturally encoded amino acid side chain (as a fusion), or indirectly connected 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 understand that the amino acid position corresponding to the position in the sequence of the specific antigen-binding polypeptide can be easily identified in the antigen-binding polypeptide or fragments of the related antigen-binding polypeptide. For example, a sequence alignment program 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 that contain one or more amino acid substitutions, additions, or deletions. The antigen-binding polypeptide of the present invention may include one or more natural amino acid modifications combined with one or more non-natural amino acid modifications. Exemplary substitutions of multiple 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 and increasing polypeptide The solubility of the peptides, the conversion of polypeptides into antagonists, etc., and these are covered 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 pyrrolelysine or selenocysteine. Other terms that can 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", "non-typical amino acid""Non-canonical amino acid" and its various hyphenated and non-hyphenated forms. The term "non-naturally encoded amino acid" also includes, but is not limited to, modification (e.g., 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- acetylglucosamine-L-serine, N- acetylglucosamine-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-threonine, N- acetyl-L-glucosamine-L-aspartic acid and O-mannosamine-L-serine. Examples of such amino acids also include examples in which the naturally occurring N- or O- bonds between the amino acid and the sugar are replaced by uncommon covalent bonds in nature, including but not limited to alkenes, oximes, and thioethers. , Amide, etc. Examples of such amino acids also include sugars that are not common in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxy-galactose, and the like. Specific examples of unnatural 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 (O-4-allyl-L-tyrosine), 4-propyl-L-tyrosine (4-propyl-L-tyrosine), Tri-O-acetyl-GlcNAc-serine (tri-O-acetyl-GlcNAcβ-serine), L-Dopa (L-DOPA), fluorinated phenylalanine (fluorinated phenylalanine), isopropyl-L- Isopropyl-L-phenylalanine, p- azido-L-phenylalanine, p- acyl-L-phenylalanine, p-phenylalanine P-benzoyl-L-phenylalanine ( p- benzoyl-L-phenylalanine), L-phosphoserine (L-phosphoserine), phosphonoserine, phosphonotyrosine, p-iodine- Phenylalanine ( p- iodo-phenylalanine), p- bromophenylalanine, p- amino-L-phenylalanine and isopropyl-L-phenylalanine -phenylalanine) and so on.

"Amine terminal modification group" refers to any molecule that can be attached to the amino terminal of a polypeptide. Similarly, "carboxy terminal modification group" refers to any molecule that can be attached to the carboxy terminus of a polypeptide. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides or proteins, such as serum albumin, or other parts that increase the serum half-life of peptides.

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 the present invention refers to a unique definable part or unit of a molecule. The terms are synonymous to some extent in the field of chemistry, and are used in the present invention to indicate a part of a molecule that performs certain functions or activities and is reactive with other molecules.

The terms "linkage", "linkage" or "linker" used in the present invention refer to groups or bonds usually formed due to chemical reactions, and are usually covalent bonds. A hydrolytically stable bond means that the bond is substantially stable in water and does not react with water at a usable pH value, including but not limited to a longer period of time under physiological conditions, or even an indefinite period. A hydrolytically unstable or degradable bond means that the bond is degradable in water or an aqueous solution (including, for example, blood). An enzymatically unstable or degradable bond means that the bond can be degraded by one or more enzymes. As understood in the art, PEG and related polymers can include degradable bonds in the polymer backbone or in the linking group between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, the ester bond formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with the alcohol group on the bioactive agent is usually hydrolyzed under physiological conditions to release the agent. Other hydrolytically degradable bonds include, but are not limited to: carbonate bonds; imine bonds produced by the reaction of amines and aldehydes; phosphate bonds formed by the reaction of alcohols with phosphoric acid groups; as the reaction product of hydrazine and aldehyde Hydrazone bond; acetal bond as the reaction product of aldehyde and alcohol; orthoester bond as the reaction product of formate and alcohol; through amine group (including but not limited to the end of polymer such as PEG) and carboxyl group of peptide The peptide bond formed; and the oligonucleotide bond formed by the phosphoramidite group (including but not limited to the end of the polymer) and the 5'hydroxyl group of the oligonucleotide. Branch linkers can be used for the antigen-binding polypeptides of the present invention.

When used in the present invention, the terms "biologically active molecule", "biologically active moiety" or "biologically active agent" refer to biological systems, pathways, and pathways that can affect organisms. Any substance with any physical or biochemical properties of molecules or interactions, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used in the present invention, biologically active molecules include, but are not limited to, those intended to be used for the diagnosis, cure, alleviation, treatment or prevention of diseases in humans or other animals, or to enhance the physical or mental health of humans or animals in other ways. Any substance. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles . The 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, antitumor agents, and cardiovascular agents. Drugs, anti-anxiety drugs, hormones, growth factor, steroid preparations, toxins from microorganisms, etc.

The anti-CD3 Fab-folate antibody of the present invention can be conjugated with molecules such as PEG to improve in vivo delivery and pharmacokinetic properties. ( Leong, SR, etc., (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 the release of agents from these linker groups include, for example, irradiation of photolabile bonds and acid-catalyzed hydrolysis. For example, U.S. Patent No. 4,671,958 includes a description of immunoconjugates that include linkers that are cleaved at target sites in the body by proteolytic enzymes of the patient's complement system. The length of the linker can be predetermined or selected according to the desired spatial relationship between the anti-CD3 antibody and the molecule to which it is linked. Given that a large number of methods for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, drugs, toxins, and other agents to antibodies have been reported, those skilled in the art will be able to determine the appropriate method to attach a given agent to an anti-CD3 antibody Or other polypeptides.

"Bifunctional polymer" refers to a polymer containing two discrete functional groups that can specifically react with other moieties (including but not limited to pendant amino acid groups) to form covalent or non-covalent bonds. A bifunctional linker having a functional group that reacts with a group on a specific biologically active component and another functional group that reacts with a group on a second biological component can be used to form a bifunctional linker that includes the first biologically active component, the bifunctional linker, and Conjugate of the second biologically active component. Many schemes and linker molecules for attaching various compounds to peptides are known. 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 by reference into the present invention. "Multifunctional polymer" refers to a polymer containing two or more discrete functional groups that can specifically react with other moieties (including but not limited to pendant amino acid groups) to form covalent or non-covalent key. The bifunctional polymer or multifunctional polymer can be any desired molecular length or molecular weight, and can be selected to provide a specific desired spacing or configuration between one of the molecules attached to the anti-CD3 antibody ( conformation).

As used in the present invention, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. The connection of the water-soluble polymer to the anti-CD3 antibody can cause changes, including but not limited to increased or adjusted serum half-life relative to the unmodified form, or increased or adjusted therapeutic half-life, 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 its own biological activity, and may be used as a linker for attaching the anti-CD3 antibody to other substances, including but not limited to one or more anti-CD3 antibodies, or one or more Biologically active molecules. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, and its mono-C1-C10 alkoxy or aryloxy derivatives (described in U.S. Patent No. 5,252,714, which is incorporated herein 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 copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, polysaccharides, cellulose and cellulose Copolymerization of derivatives (including but not limited to methyl cellulose and carboxymethyl cellulose), starch and starch derivatives, polypeptides, polyalkylene glycols and their derivatives, polyalkylene glycols and their derivatives Polyvinyl ether and α-β-poly[[2-hydroxyethyl)-DL-aspartic acid, etc., or mixtures thereof. Examples of such water-soluble polymers include, but are not limited to, polyethylene glycol and serum albumin.

As used in the present invention, the term "polyalkylene glycol (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, the "polyalkylene glycol" may be about 5K to 50K or between 5K and 50K. For example, other exemplary embodiments are listed in a catalog of commercial suppliers, such as the catalog of Shearwater Corporation "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). As used in the present invention, polyethylene glycols having molecular weights of 5kDa, 10kDa, 20kDa, etc. are referred to as "5K PEG", "10K PEG", "20K PEG, etc." in the present invention, respectively.

As used in the present invention, the term "modulated serum half-life" refers to the positive or negative change in the circulating half-life of a modified anti-CD3 antibody relative to its unmodified form. The serum half-life is determined by taking blood samples at different time points after administration of the anti-CD3 antibody and determining the concentration of the molecule in each sample. The correlation between serum concentration and time allows calculation of serum half-life. It is expected that the serum half-life will increase by at least about a two-fold, but a smaller increase may also be useful, for example where it can achieve 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 in the present invention, the term "modulated therapeutic half-life" refers to the positive or positive half-life of a therapeutically effective amount of an anti-CD3 antibody or an anti-CD3 antibody containing a modified biologically active molecule relative to its non-modified form. Negative change. The treatment half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. The increase in the treatment half-life ideally enables a specific beneficial dosing regimen, a specific beneficial total dose, or the avoidance of unwanted effects. In one embodiment, the increased treatment half-life is due to increased potency, increased or decreased binding of the modified molecule to its target, or increased or decreased 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 associated with it in its natural state. It can be in a homogeneous state. The separated substance can be in a dry or semi-dried state, or in a solution, including but not limited to an aqueous solution. Purity and homogeneity are usually 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 preparation, is substantially purified. In particular, the isolated gene is separated from the open reading frame flanking the gene and encoding a protein other than the gene of interest. The term "purified" means that the nucleic acid or protein produces essentially a band in the electrophoresis gel. In particular, this means that the purity of the nucleic acid or protein is at least 85%, the purity is at least 90%, the purity is at least 95%, and the purity is at least 99% or higher.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically defined otherwise, the term also refers to oligonucleotide analogs (oligonucleotide analogs), which include PNA (peptidonucleic acid, peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate) Ester, amino phosphate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants (including but not limited to degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. 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 mixed bases and/or deoxyinosine residues (Batzer Et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994 ).

The terms "polypeptide", "peptide" and "protein" are used interchangeably in the present invention and refer to a polymer of amino acid residues. That is to say, the description of polypeptides is also applicable to the description of peptides and the description of proteins, and vice versa. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used in the present invention, the term encompasses amino acid chains of any length, including full-length proteins (ie antigens), in which the amino acid residues are connected 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, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamine, glycine, Histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and Pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (that is, the α carbon combined with hydrogen, carboxyl, amine and R groups), such as homoserine, leucine , Methionine sulfoxide (methionine sulfoxide), methionine methyl sulfonium (methionine methyl sulfonium). Such analogs have modified R groups (for example, ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids.

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

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With regard to specific nucleic acid sequences, "conservatively modified variants" refer to those nucleic acids that encode the same or substantially the same amino acid sequence, or when the nucleic acid does not encode the amino acid sequence, it refers to the substantially same 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 encode alanine. Therefore, at each position where alanine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations", which are a type of conservatively modified variations. Every nucleic acid sequence encoding a polypeptide in the present invention also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG and TGG, which is usually the only codon for methionine, and TGG is usually the only codon for tryptophan) can be modified to produce a function The same molecule. Therefore, every silent variation of the nucleic acid encoding the polypeptide is implicit in every said 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 that change, add, or delete a single amino acid or a small part of the amino acid in the coding sequence Is a "conservatively modified variant" in which the change causes the amino acid to be replaced by 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 the supplement of the polymorphic variants, interspecies homologs and alleles of the present invention, and do not exclude the polymorphic variants and interspecies 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), glutamine (E); 3) Aspartic acid (N), glutamic acid (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), Methionine (M), Valine (V); 6) Amphetamine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine ( T); and 8) Cysteine (C), Methionine (M); (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman &Co.; 2nd edition (December 1993)) ).

In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage refers to two or more sequences or subsequences that are identical. When using one of the following sequence comparison algorithms or measuring by manual alignment and visual inspection, compare and align on the comparison window or designated area to obtain maximum correspondence, if the sequence has a certain percentage of the same amino acid Residues or nucleotides (ie, about 60% identity within a specified region, optionally about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity性), then the sequence is "substantially the same." This definition also refers to the complement of the test sequence. The identity can exist in a region of at least about 50 amino acids or nucleotides in length, or in a region of 75-100 amino acids or nucleotides in length, or in the entire sequence or when not specified. Polynucleotide or polypeptide.

For sequence comparison, a sequence is usually used as a reference sequence and compared with a test sequence. When using the sequence comparison algorithm, input the test sequence and the reference sequence into the computer, specify the sub-sequence coordinates if necessary, and specify the sequence algorithm program parameters. Default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the percent sequence identities (the percent sequence identities) of the test sequence relative to the reference sequence based on the program parameters.

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

As used in the present invention, the term "effective amount" refers to the amount of (modified) non-natural amino acid polypeptide administered, which will alleviate one or more symptoms of the disease, disorder or disorder being treated to some extent. The composition containing the (modified) non-natural amino acid polypeptide of the present invention can be administered for preventive, enhancing and/or therapeutic treatment.

The term "enhancement" refers to increasing or prolonging the potency or duration of a desired effect. Therefore, with regard to enhancing the effect of a therapeutic agent, the term "enhancement" refers to the ability to increase or prolong the effect of other therapeutic agents on the system in terms of effectiveness or duration. As used in the present invention, "enhancing-effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system. When used in a patient, the effective dosage will depend on the severity and course of the disease, disorder or condition, previous treatment, the patient's health status and response to the drug, and the judgment of the attending physician.

As used in the present invention, the term "modified" refers to the presence of post-translational modifications on the polypeptide. The form "(modified)" term means that the polypeptide in question is optionally modified, that is, the polypeptide in question may be modified or unmodified.

The terms "post-translationally modified" and "modified" refer to any modification of a natural or unnatural amino acid that occurs after the amino acid is incorporated into the polypeptide chain. For example only, the term encompasses co-translational in vivo modifications, post-translational in vivo modifications, and post-translational in vitro modifications.

In prophylactic or therapeutic applications, a composition containing (modified) non-natural amino acid polypeptide is administered to an already suffering disease, disorder, or disorder in an amount sufficient to cure or at least partially prevent the symptoms of the disease, disorder, or disorder Of patients. Such an amount is defined as a "prophylactically effective amount" or a "therapeutically effective amount" and will depend on the severity and course of the disease, disorder or condition, previous treatment, the patient’s health status and response to the drug, and the attending physician’s judge. It is believed that the determination of this therapeutically effective amount through routine experiments (such as a dose-escalation clinical trial) is completely within the technical scope of the art.

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

Unless otherwise specified, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the technical scope of the art are used.

According to the present invention, more specific implementations are proposed below.

introduction:

The inventors have developed bispecific antibodies that contain 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 can 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. The anti-CD3 antibody, fragment or variant comprises an amino acid that has 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 in the heavy chain of the Fab (according to Kabat numbering), and positions 157, 172, and 205 in the light chain (According to Kabat numbering). Bispecific antibodies may comprise amino acids that have been engineered to have one or more non-naturally encoded amino acids on the heavy chain (K129; Kabat numbering) and light chain (L157; Kabat numbering) of the Fab, such as p-acetyl- Phenylalanine (pAF) anti-CD3 Fab. The bispecific antibody may be composed of an anti-CD3 Fab that has been engineered to have one or more non-natural codes on the heavy chain (K129; Kabat numbering) and light chain (L157; Kabat numbering) of the Fab The amino acid, such as p-acetyl-phenylalanine (pAF). In some embodiments of the present 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 one or more anti-CD3 antibodies. Conjugation of an unnatural amino acid (e.g. pAF) to produce, for example, one or two PEG and/or folic acid molecules that are stably conjugated to an anti-CD3 Fab. Adding one or more PEG molecules (such as 5K, 10K, or 20K PEG) can significantly improve the pharmacokinetic properties of bispecific antibodies, while maintaining specific cytotoxicity against FOLR1 expressing cells in vitro and in vivo. It was observed that tumor-promoting macrophages (M2) and MDSC cells were preferentially consumed by the pegylated anti-CD3 Fab-folate composition disclosed in the present invention. The results indicate that improved pharmacokinetic properties will require less frequent dosing, and administration using infusion pumps can be avoided. As used in the present invention, the terms "PEG-folate" or "folate-PEG" and "bisPEG-difolate" or "difolate-bisPEG" are used interchangeably.

Antibodies, antibody fragments and variants:

The antibodies or antibody fragments or variants of the present invention can be human, humanized, engineered, non-human and/or chimeric antibodies or antibody fragments. The antibodies or antibody fragments or variants provided by the present invention may contain two or more amino acid sequences. The first amino acid sequence may comprise a first antibody chain and the second amino acid sequence may comprise a second antibody chain. The first antibody chain may include a first amino acid sequence and the second antibody chain may include a second amino acid sequence. The antibody chain can 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 the antibody light chain. As a non-limiting example, the antibody provided by the present invention comprises a heavy chain or a fragment or variant thereof, and a light chain or a fragment or variant thereof. The 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 a combination thereof. Chemical linkers include linkers via non-natural amino acids. Chemical linkers include linkers via one or more non-natural amino acids. The chemical linker may include a chemical conjugate. A peptide linker includes 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 More, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. The peptide linker can be part of any antibody, including antibody domains, such as variable domains, CH1, CH2, CH3, and/or CL domains. In one embodiment, the heavy chain and the light chain are connected via a peptide linker, for example. In some cases, the heavy chain and light chain are connected, for example, by one or more disulfide bonds.

The antibodies, antibody fragments and antibody variants of the present disclosure can interact or engage with 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, white blood cells, lymphocytes, macrophages, red blood cells, platelets, mesophils Sex 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 in the present invention can bind to cell surface receptors. The antibodies or antibody fragments disclosed in the present invention can bind to folate receptors. The antibodies or antibody fragments disclosed in the present invention 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) or its analogues 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 in the present invention are anti-CD3 antibodies or antibody fragments or variants thereof. In certain embodiments, the anti-CD3 antibodies or antibody fragments or variants disclosed in the present invention can be humanized. The anti-CD3 antibodies or antibody fragments or variants disclosed in the present invention include but are not limited to CD3 analogs, isoforms, analogs, fragments or hybrids. The anti-CD3 antibodies or antibody fragments or variants of the present invention include but are not limited to Fv, Fc, Fab and (Fab') 2, single chain Fv (scFv), diabody, trisomy, tetrasomy, bifunctional hetero Combine 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 antibody or antibody fragment or variant of the present invention comprises the sequence of SEQ. ID. NO: 1 to 62. The antibody, fragment or variant of the present invention may be an anti-CD3 Fab antibody, fragment or variant. The antibodies, fragments or variants of the invention may comprise one or more anti-CD3 Fabs. The 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, the anti-CD3 antibody is composed 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. 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 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 one of 39, 58, 59, 60, 61, and 62.

The invention also discloses an anti-CD3 bispecific antibody containing non-natural 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, trisomy, Tetrabody, bifunctional hybrid antibody, CDR1, CDR2, CDR3, CDR combination, variable region, framework region, constant region, heavy chain, light chain, alternative scaffold non-antibody molecule, bispecific antibody, etc. In one embodiment, the anti-CD3 bispecific antibody or antibody fragment or variant is an anti-CD3 Fab bispecific antibody, fragment or variant comprising one or more non-naturally encoded amino acids. The anti-CD3 Fab bispecific antibody or antibody fragment or variant of the present 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 is composed 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 example, the bispecific antibody, fragment or variant disclosed in the present invention specifically binds to 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. The bispecific antibody may be a humanized antibody.

Table 1: Novel amino acid sequences of humanized anti-CD3 variants with and without HC-DKTHT extension, in which underscores are used in the heavy and light chains where the unnatural amino acid pAF is incorporated. All sequences in the table below are also disclosed, where pAF is replaced by any other non-natural amino acid.

Non-natural amino acid:

The invention provides anti-CD3 antibodies, antibody fragments or variants comprising at least one non-naturally encoded amino acid. The introduction of at least one non-naturally encoded amino acid into an anti-CD3 antibody may allow the application of conjugation chemistry, which participates in a specific chemical reaction with one or more non-naturally encoded amino acids, without interacting with the 20 commonly present amines. Base acid reaction.

In some embodiments disclosed in the present invention, it is an anti-CD3 antibody containing one or more non-naturally encoded amino acids. The one or more non-natural amino acids may be encoded by a codon that does not encode one of the 20 natural amino acids. One or more non-natural amino acids can be encoded by a nonsense codon (stop codon). The stop codon can be an amber codon. Amber codons can contain UAG sequences. The stop codon can be an ocher codon. Ochre codons can contain UAA sequences. The stop codon can be an opal or umber codon (opal or umber codon). Opal or umber codons can contain UGA sequences. One or more unnatural amino acids can be encoded by four-base codons.

One or more unnatural amino acids may include, but are not limited to, pAz (pAz), pBpF, pPrF, p-Iodoamphetamine Acid (pIF), p-cyanophenylalanine (pCNF), p-carboxymethamphetamine (pCmF), 3-(2-naphthyl)alanine (NapA), p-borate phenylalanine (pBoF), o-nitro Phenylalanine (oNiF), (8-quinolin-3-yl)alanine (HQA) and (2,2'-bipyridin-5-yl)alanine (BipyA). One or more unnatural amino acids can be β-amino acids (β3 and β2), peramino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives , Ring-substituted phenylalanine and tyrosine derivatives, linear core amino acid, diamino acid, D-amino acid, N-methylamino acid or a combination 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-nitro -L-tyrosine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine and p-isopropyl-L-phenylalanine; 2) has an aryl azide that can be crosslinked by light 3) Amino acids with unique chemical reactivity, including acetyl-L-phenylalanine and meta-acetyl-L-phenylalanine, O-allyl -L-tyrosine, O-(2-propynyl)-L-tyrosine, p-ethylthiocarbonyl-L-phenylalanine and p-(3-oxobutyryl)-L-phenylalanine; 4 ) Heavy atom-containing amino acids used for phasing in X-ray crystallography, including p-iodo-L-phenylalanine and p-bromo-L-amphetamine; 5) Redox active amino acid dihydroxy-L-amphetamine Acid; 6) Glycosylated amino acids, including bN-acetylglucosamine-O-serine and aN-acetylgalactosamine-O-threonine; 7) with naphthyl and dansyl 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 ) Phosphotyrosine mimetic p-carboxymethyl-L-phenylalanine; 10) Glutamine homologue homoglutamic acid; and 11) 2-aminocaprylic acid. In one embodiment, the non-natural amino acids are N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-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-threonine, N-acetyl-L-glucosamine-L-aspartic acid and O-mannosamine-L-serine. Examples of such amino acids also include the following examples: wherein the naturally occurring N- or O- bonds between the amino acid and the sugar are replaced by covalent bonds that are not common in nature, including but not limited to olefins, oximes, sulfur Ether, amide, etc. Examples of such amino acids also include sugars that are not commonly found 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 Group) alanine (L-3-(2-naphthyl)alanine), 3-methyl-phenylalanine (3-methyl-phenylalanine), O-4-allyl-L-tyrosine, 4-propyl -L-Tyrosine (4-propyl-L-tyrosine), Tri-O-Acetyl-GlcNAc''-serine, L-Dopa (L-DOPA), Fluorinated Phenylalanine, Isopropyl-L -Phenylalanine, p-azido-L-phenylalanine, p-aniline-L-phenylalanine, p-anisyl-L-phenylalanine, L-phosphoserine, phosphoserine, phosphatidyl Amino acid, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine and isopropyl-L-phenylalanine, etc. Other 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 Number: PCT/US2012/039472 , PCT/US2012/039468, PCT/US2007/088009, PCT/US2009/058668, PCT/US2007/089142, PCT/US2007/088011, PCT/US2007/001485, PCT/US2006/049397, PCT/US2006/047822 and PCT In /US2006/044682, the entire content of each article in the document is incorporated into the present invention by reference. In one embodiment, the one or more non-natural amino acids may be p-acetylphenylalanine (pAF).

In certain embodiments of the invention, the anti-CD3 antibody with at least one unnatural amino acid includes at least one post-translational modification. In one embodiment, the at least one post-translational modification includes attaching the following molecules containing the second reactive group to at least one containing the first reaction using chemical methods known to those of ordinary skill in the art to be suitable for the specific reactive group Groups of non-natural amino acids, the molecules include but are not limited to water-soluble polymers, polyethylene glycol derivatives, drugs, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, biologically active agents, Small molecules, or any combination of the above or any other desired compounds or 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), and the second reactive group The group is the azido moiety and utilizes [3+2] cycloaddition chemistry. In another example, the first reactive group is an azido 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 present invention, at least one non-natural amino acid (including but not limited to non-natural amino acid containing a ketone functional group) is used, which includes at least one post-translational modification, wherein The at least one post-translational modification comprises a sugar moiety. In certain embodiments, the post-translational modification is performed in vivo in eukaryotic or non-eukaryotic cells. In other embodiments, the post-translational modification is performed in vitro. In another embodiment, the post-translational modification is performed in vitro and in vivo.

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

In some embodiments disclosed in the present invention, non-natural amino acids are position-specifically incorporated into antibodies, antibody fragments or variants. In one embodiment, the non-natural amino acid is position-specifically incorporated into the anti-CD3 antibody, antibody fragment or variant. Methods of incorporating non-natural amino acids into molecules such as proteins, polypeptides or peptides are disclosed in U.S. Patent Nos.: 7,332,571; 7,928,163; 7,696,312; 8,008,456; 8,048,988; 8,809,511; 8,859,802; 8,791,231; 8,476,411; or 9,637,411 (all of each The content is incorporated by reference into the present invention), as well as the embodiments of the present invention. One or more non-natural amino acids can be incorporated by methods known in the art. For example, cell-based or cell-free systems can be used, and auxotrophic strains can also be used instead of engineered tRNA and synthetase. 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/ As disclosed in 046618, each of said documents is incorporated into the present invention in its entirety by reference. 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. 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. Mutation of one or more nucleotides in the nucleotide sequence encoding the antibody or antibody fragment or variant may include changing a codon encoding an amino acid to a nonsense codon. Incorporating one or more unnatural amino acids into the 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 in the antibody or antibody fragment or variant Generate one or more amber codons (amber codons). One or more unnatural amino acids can be incorporated into the antibody or antibody fragment or variant in response to an amber codon. One or more unnatural amino acids can be incorporated into the antibody or antibody fragment or variant specifically. The incorporation of one or more non-natural amino acids into antibodies or antibody fragments or variants may include one or more genetic codes with orthogonal chemical reactivity relative to the typical twenty amino acids The non-natural amino acids of sulphate can be used to position-specifically modify biologically active molecules or targeting agents. Incorporating one or more unnatural amino acids may include using a tRNA/aminoacyl-tRNA synthetase pair (a tRNA/aminoacyl-tRNA synthetase pair) in response to one or more amber nonsense codons (amber nonsense codon). ) Position-specifically incorporate one or more non-natural amino acids into a defined position in a biologically active molecule or targeting agent. Other methods for incorporating non-natural amino acids include, but are not limited to, the methods disclosed in: Chatterjee et al., a universal platform for single and multiple non-natural amino acid mutagenesis in E. coli, (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 non-natural amino acids can be produced by the selective reaction of one or more natural amino acids. The selective reaction can be mediated by one or more enzymes. In a non-limiting example, the selective reaction of one or more cysteines with formylglycine (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. The chemical reaction to form the linker may include a bioorthogonal reaction. The chemical reaction to form the linker may include click chemistry. See, for example, WO2006/050262, which is incorporated into the present invention in its entirety by reference.

Bioactive molecules/reagents:

The present invention discloses an anti-CD3 antibody or an antibody fragment or variant thereof, which comprises 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 reagents, non-peptide compounds, drugs, second proteins or polypeptides or polypeptide analogs or derivatives, antibodies or antibody fragments or variants, second biologically active agents, targeting agents, or Any combination of the above or any other desired compound or substance. In one embodiment, the bioactive agent involves the recruitment of cytotoxic T cells to cells, including but not limited to cancer cells or tumor cells. In one embodiment, the biologically active 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 biologically active molecule is folic acid or a derivative or analogue thereof. Biologically active molecules can 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, neurokinin 1 receptor, melanocortin 1 receptor , Neurotensin receptor, neuropeptide Y receptor and C-type lectin like molecule 1, receptor, co-receptor, transmembrane protein or cell marker or cell surface protein. Biologically active molecules can bind to target cells. The biologically active molecule can bind to cell surface proteins or cell surface markers or cell surface molecules on the cell. Biologically active molecules can bind to cell surface molecules on cells including but not limited to cancer cells or tumor cells or immunosuppressive cells. The cell surface molecule may be a folate receptor molecule. The biologically active molecule may be an agent that binds to prostate specific membrane antigen (PSMA), such as but not limited to DUPA or its analogs or derivatives. Biologically active molecules or agents can bind to cells that overexpress or highly express cell surface markers, proteins, or receptors.

In an embodiment, the biologically active molecule may be folate or a folate ligand or an analog or derivative thereof. Biologically active molecules can bind to folate receptor protein (FR). Such biologically active molecules can be N-(4-{[(2-amino-4-oxo-1,4-dihydropterin-6-yl)methyl]amino}benzyl)- L-glutamic acid (folic acid), or its analogs or derivatives. Its analogs can be based on folic acid and retain the FR-bound part. Folic acid analogs can retain a large part of the structure of folic acid. In addition, since the folate analog is conjugated to a linker or antibody or antibody fragment or variant, the folate analog may be a slightly modified form of folate. For example, the folate analog can be slightly modified due to the conjugation of the folate carboxyl group to the linker or antibody or antibody fragment or variant. In addition, due to the conjugation of folic acid to linkers or antibodies or antibody fragments, folic acid can be slightly modified, but still retains its FR binding properties. In one embodiment, the folate molecule targets folate receptor alpha. In one embodiment, the folate molecule targets folate receptor β. In one embodiment, folic acid or folate is used as a bioactive molecule or targeting agent that binds to folate receptor (FR) antigen, which is overexpressed or highly expressed on FR+ cell lines. In one embodiment, the cell is a cancer cell or an immunosuppressive cell, but it is not limited thereto.

The biologically active molecule or agent can be position-specifically linked to one or more unnatural amino acids of the 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 non-natural amino acids. Chemical linkers include linkers via one or more non-natural amino acids. Chemical linkers can include chemical conjugates. A peptide linker includes 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 More, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more amino acids. The peptide linker can be part of any antibody, including antibody domains, such as variable domains, CH1, CH2, CH3, and/or CL domains. The antibody or antibody fragment or variant can be conjugated to a biologically active molecule. Antibodies or antibody fragments or variants can be conjugated to biologically active molecules via chemical and/or peptide linkers. In one embodiment, the present invention provides anti-CD3 antibodies or antibody fragments or variants conjugated to one or more biologically active molecules or agents. In one embodiment, the one or more biologically active molecules or agents are one or more small molecules. In one embodiment, the present 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 an anti-CD Fab antibody via a chemical and/or peptide linker.

PEG linker/ligand/conjugate:

The anti-CD3 antibody or antigen-binding polypeptide and the small molecule can be joined by linkers, polymers or covalent bonds. The linker, polymer, or small molecule itself can contain functional groups that are not reactive with 20 common amino acids. The linker or polymer may be a bifunctional linker or polymer. The bifunctional linker or polymer is a branched linker or polymer. One or more bonds involved in joining an anti-CD3 antibody or antigen-binding polypeptide to a 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 the controlled release of the antigen-binding polypeptide or other molecule. Those skilled in the art can produce various small molecules through chemical means, separation as natural products or other means.

The present invention discloses an anti-CD3 antibody or antibody fragment or variant, which comprises 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 containing a non-naturally encoded amino acid can be linked to two water-soluble polymers, such as two polyethylene glycol (PEG) molecules or ligands. An antibody or antibody fragment or variant comprising a non-naturally encoded amino acid and one or more biologically active molecules can be linked to one or more water-soluble polymers, such as polyethylene glycol (PEG) molecules or linkers. In 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 biologically active molecule or a water-soluble polymer or a conjugate comprising a biologically active molecule and a water-soluble polymer. The method may include conjugating one or more linkers to a biologically active molecule to produce a biologically active molecule-linker intermediate, and conjugating the intermediate to the antibody or antibody fragment. The method can include conjugating one or more linkers to a PEG molecule to 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 the antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate, and co-coating the antibody-linker intermediate or antibody fragment-linker intermediate Conjugation to another biologically active molecule, or a water-soluble polymer, or a conjugate comprising a biologically active molecule and a water-soluble polymer. The method disclosed in the present invention may include conjugating one or more linkers with one or more antibodies or antibody fragments, one or more biologically active molecules or a combination thereof to produce one or more intermediates, such as antibody-linker intermediates , Antibody fragment-linker intermediate and/or bioactive molecule antibody conjugate-linker intermediate. The method can include conjugating the first linker to the antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate. The method may include conjugating the linker with the biologically active molecule to produce a biologically active molecule-linker intermediate.

The method of producing the bispecific anti-CD3 antibody conjugate of the present invention may include (a) conjugated a first linker to an antibody or antibody fragment comprising one or more unnatural amino acids incorporated into the antibody or antibody fragment , (B) conjugate a second linker with a biologically active molecule to produce a biologically active molecule-linker intermediate; and (c) link the two intermediates together to produce an anti-CD3 antibody-biologically active molecule conjugate; biological The active molecule can be a small molecule, including but not limited to folic acid or DUPA molecules or their analogs or derivatives. In certain embodiments, the method for producing the bispecific anti-CD3 antibody conjugate of the present invention may include: (a) combining a first linker with one or more unnatural amine groups incorporated into the antibody or antibody fragment Conjugation of an acidic antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate; (b) conjugate a second linker with a biologically active molecule to produce a biologically active molecule-linker intermediate; and (c ) The two intermediates are linked together to produce 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 their analogs or derivatives. The method for producing the bispecific anti-CD3 Fab antibody-folate conjugate of the present invention may include: (a) combining a first linker with an antibody containing one or more unnatural amino acids incorporated into the antibody or antibody fragment; Conjugation of a CD3 Fab antibody or antibody fragment to produce an antibody-linker intermediate or antibody fragment-linker intermediate; (b) conjugate 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 Fab antibody-bioactive molecule conjugate; the bioactive molecule includes one or more folate or DUPA molecules or analogs or derivatives; the linker includes one or Multiple chemical and/or peptide linkers; the linker comprises one or more PEG molecules. 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 more. The average molecular weight of PEG molecules is 5K, 10K or 20K.

The conjugation of one or more linkers to the antibody or antibody fragment or biologically active molecule can occur simultaneously. The conjugation of one or more linkers to the antibody or antibody fragment or biologically active molecule can occur sequentially. The conjugation of one or more linkers to the antibody or antibody fragment or biologically active molecule can be carried out in a one-step method such as enzymatic conjugation or a chemical step or process or reaction. The conjugation of one or more linkers to the antibody or antibody fragment or biologically active molecule can be carried out in a two-step method such as two enzymatic conjugation or two chemical steps or two processes or two reactions. The conjugation of one or more linkers to the antibody or antibody fragment or biologically active molecule can be carried out 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, conjugating intermediates with antibodies or antibody fragments or biologically active molecules or water-soluble polymers may include oxime chemistry to form oxime bonds and/or click chemistry ( click chemistry). The antibody or antibody fragment may contain one or more unnatural amino acids. Linking the antibody or antibody fragment to the intermediate can include the formation of an oxime between the non-natural amino acid and the linker intermediate. Conjugating the linker with the antibody or antibody fragment or biologically active molecule or water-soluble molecule may include an ionic bond, covalent bond, non-covalent bond 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 biologically active molecules or water-soluble molecules is known in the art. See, for example, Roberts et al., Advanced Drug Delivery Reviews 54:459-476 (2002).

Conjugating one or more linkers to the antibody or antibody fragment and/or biologically active molecule may include forming one or more oximes between the linker and the antibody or antibody fragment or biologically active molecule. Conjugating one or more linkers to the 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 the antibody or antibody fragment and/or biologically active molecule can 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 the antibody or antibody fragment and/or biologically active molecule can 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 biologically active molecule.

Conjugating one or more linkers to the antibody or antibody fragment may include position-specifically conjugating one or more linkers to the antibody or antibody fragment. Position-specific conjugation can include linking one or more linkers to the non-natural amino acid of the antibody or antibody fragment. Linking one or more linkers to the non-natural amino acid of the antibody or antibody fragment can include the formation of an oxime. Linking one or more linkers to the non-natural amino acid of the antibody or antibody fragment may include the formation of sulfides. As a non-limiting example, linking one or more linkers to the non-natural amino acid of the antibody or antibody fragment may include reacting the hydroxylamine of one or more linkers with the 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 the non-natural amino acid of the antibody or antibody fragment may include reacting the bromo derivative of one or more linkers with the thiol of the amino acid. The amino acid may be a non-natural amino acid.

One or more PEGs or linkers may contain disulfide bonds that connect two cysteine residues using conjugation chemistry known to those skilled in the art. (See, for example, ThioBridge™ technology, Abzena). Two or more PEG molecules or linkers may comprise a maleimide bridge 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 example, two or more C-terminal PEG molecules can be covalently linked or cross-linked between the heavy and light chains of the antibody or antibody fragment. Such PEG conjugates or linkers are described in the present invention.

In one embodiment, the present invention discloses a folate-PEG linker comprising one or more folic acid and one or more PEG molecules. The one or more PEG molecules may be 5kDa, 10kDa, 15kDa, 20kDa 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 a molecular weight of 0.1 kDa to 50 kDa. 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 1 kDa to 100 kDa, or 1 kDa to 50 kDa. PEG molecules are well known in the art, see, for example, Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001).

In certain embodiments, the present invention discloses anti-CD3 Fab-folate PEGylated conjugates comprising one or more PEG molecules. In certain embodiments, the present 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 separately attached to the heavy and light chains of the antibody or antibody fragment. In an embodiment, two or more C-terminal PEG molecules can be covalently attached or cross-linked between the heavy and light chains of the antibody or antibody fragment. In one embodiment, two or more PEG molecules may be covalently attached or crosslinked via a maleimine bridge connecting two cysteine residues.

Pegylation can be used to improve the pharmacokinetics of the composition and regulate cytotoxicity. The PEGylation of protein can increase its serum half-life by delaying renal clearance, because the PEG moiety adds a considerable hydrodynamic radius to the protein. The covalent attachment of hydrophilic polymer polyethylene glycol (abbreviated as PEG) is for many biologically active molecules (including proteins, peptides, especially hydrophobic molecules) to increase water solubility, bioavailability, prolong serum half-life, and extend Methods of treatment half-life, adjustment of immunogenicity, adjustment of biological activity, or extension of cycle time. PEG has been widely used in drugs, artificial implants, and other applications where biocompatibility, non-toxicity, and non-immunogenicity are important. Preferably, pegylation does not change or only minimally changes the activity of the biologically active molecule. Preferably, the increase in half-life is greater than any decrease in biological activity. Rader et al. described in Proc Natl Acad Sci US A. 2003.04.29; 100(9):5396-400, which is incorporated by reference into the present invention. Provides a method of effector function and prolonging the serum half-life. The described complex is a combination of mAb 38C2 (a catalytic antibody that mimics natural aldolase) and an integrin targeting Arg-Gly-Asp peptidomimetic via reactive lysine residues on the antibody. Reversible covalent bonds between ketone derivatives are produced. In addition to increasing the peptidomimetic half-life, the complex has also been shown to selectively retarget antibodies to the surface of cells expressing integrins αvβ3 and αvβ5.

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 a 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 achieved via a 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 folate derivative via a side chain of the non-naturally encoded amino acid. In one embodiment, an anti-CD3 antibody comprising a non-naturally encoded amino acid is attached to a water-soluble polymer such as polyethylene glycol (PEG), wherein the attachment is achieved via a side chain of the non-naturally encoded amino acid. In one example, an anti-CD3 antibody comprising a non-naturally encoded amino acid is attached to a water-soluble polymer derivative such as a polyethylene glycol (PEG) derivative, wherein the attachment is via the side of the non-naturally encoded amino acid. Chain realization. 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. Connect 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, and 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 can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 kDa. The molecular weight of polyethylene glycol may be about 1 kDa to about 25 kDa, or 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 5 kDa or 10 kDa or 20 kDa. The molecular weight of polyethylene glycol can be 5 kDa.

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

in:

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-, -(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–,

Wherein 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 where

Y is selected from hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, ketone-amine, ketone- Alkynes, alkynes, cycloalkynes, and en-diones.

In one embodiment, B is a substituted divalent heterohydrocarbyl residue. In one embodiment, the substituent includes one or more carboxyl, ketone and/or amide functional groups. In one embodiment, the heteroatom is 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 substituent includes one or more carboxyl, ketone and/or amide functional groups. In one embodiment, the heteroatom is 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 1-20, or 1-10, or 1-5.

在一實施例中，F具有以下結構：

In one embodiment, F has the following structure:

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

在一實施例中，雙官能水溶性聚合物-葉酸接頭具有以下結構：

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

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

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

In one embodiment, Z has the following structure:

in:

J is optional, and when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower alkylene Group, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene (lower heterocycloalkylene), substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene heteroarylene), alkarylene, substituted alkylene, aralkylene or substituted aralkylene;

G is optional, and when present, is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, Substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- (where k is 1, 2 or 3), -S(O)k (alkylene or substituted alkylene)-, -C(O)-, -C(O) -(Alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(alkylene or substituted alkylene)- , -CSN(R')-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene) -, -N(R')C(O)O-, -S(O)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')=NN(R')-, -C(R')=NN=, -C(R')2-N=N- and -C(R')2-N(R')-N (R')-, where each R'is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;

R1 is H, an amino protecting group, 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 two R3 groups optionally form cycloalkyl or heterocycloalkyl.

In one embodiment, Z has the following structure:

Where J, G, R1, R2, R3 and R4 are as defined above, and

Where D has the following structure:

Where 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, alkylaryl, Substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene) -ON(R'')2, -(alkylene or substituted alkylene) Alkyl) -C(O)SR'', -(alkylene or substituted alkylene) -SS-(aryl or substituted aryl), -C(O)R'', -C (O)2R" or -C(O)N(R")2, wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkane Oxy, 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 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 T3 is O or S, R'' cannot be a halogen;

Each R" is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

m and p are 0, 1, 2 or 3, provided that at least one of m or p is not 0;

、

、

、

、

、

、

、

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；M2 is

,

,

,

,

,

,

,

or

, Where (a) represents a bond with the B group and (b) represents a bond with the corresponding position in the heterocyclic group;

、

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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；M ₃ is

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,

or

, Where (a) represents a bond with the B group and (b) represents a bond with the corresponding position in the heterocyclic group;

、

、

、

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；M ₄ is

,

,

,

or

, Where (a) represents a bond with the B group and (b) represents a bond with the corresponding position in 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, aryl amide, Alkyl halides, alkyl amines, alkyl sulfonic acids, alkyl nitro groups, thioesters, sulfonyl esters, halosulfonyl groups, nitriles, alkyl nitriles and nitro groups;

q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; and each R16 is independently selected from hydrogen, halogen, alkyl, NO2, CN, and substituted alkyl .

The present invention provides an efficient method for selectively modifying proteins with PEG derivatives. The method includes responding to selector codons to convert non-genetically encoded amino acids (including but not limited to those containing 20 naturally incorporated amino acids). Functional groups or substituents (including but not limited to ketone, azide, or acetylene moieties) not found in amino acids) are selectively incorporated into the protein and then those amino acids are modified with suitable reactive PEG derivatives. Once incorporated, chemical methods known to those of ordinary skill in the art can be used to modify the amino acid side chains to suit the specific functional groups or substituents present in the naturally encoded amino acid. Various known chemical methods are suitable for the present invention to incorporate water-soluble polymers into proteins. Such methods include, but are not limited to, the Huisgen [3+2] cycloaddition reaction (see, for example, Padwa, A. Comprehensive Organic Synthesis, Volume 4, Trost, BM, Pergamon eds, Oxford, pages 1069-1109, 1991; and Huisgen, R. 1,3-Dipolar Cycloaddition Chemistry, Padwa, A. Ed., Wiley, New York, pp. 1-176, 1984), which respectively include but are not limited to acetylene or azide derivatives.

Because the Huisgen [3+2] cycloaddition method involves a cycloaddition rather than a nucleophilic substitution reaction, 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 carried out with excellent regioselectivity (1,4> 1,5) under aqueous conditions at room temperature. (See, for example, 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). The molecules that can be added to the protein of the present invention through a [3+2] cycloaddition reaction actually include any molecule with suitable functional groups or substituents, including but not limited to azido or acetylene derivatives. These molecules can be added to non-natural amino acids with ethynyl groups (including but not limited to p-propargyloxyphenylalanine) or non-natural amino acids with azido groups (including but not limited to p-azido -Phenylalanine).

The five-membered ring produced by the Huisgen [3+2] cycloaddition reaction is usually irreversible in a reducing environment, and can be stable for a long time without hydrolysis in an aqueous environment. Therefore, the active PEG or PEG derivatives of the present invention can be used to modify the physical and chemical properties of various substances under highly demanding aqueous conditions. More importantly, because the azide and acetylene moieties are specific to each other (for example, they will not react with any of the 20 common genes encoding amino acids), they can be used with extremely high selectivity. The protein is modified in one or more specific locations.

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. The PEG polymer derivative containing an acetylene moiety has high selectivity for conjugation with the azide moiety that has been selectively introduced into the protein in response to the selector codon. Similarly, PEG polymer derivatives containing azide moieties have high selectivity for conjugation with acetylene moieties that have been selectively introduced into proteins in response to selector codons.

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

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

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

anti- CD3 Antibody activity and anti CD3 Measurement of the affinity of an antibody to its antigen or binding partner :

Standard in vitro or in vivo assays can be used to determine the activity of anti-CD3 antibodies. For example, cells or cell lines that bind anti-CD3 antibodies (including but not limited to cells containing natural 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 the BIAcore™ biosensor (Pharmacia) or Octet (ForteBio) to measure.

No matter which method is used to produce anti-CD3 antibodies, the biological activity of anti-CD3 antibodies must be determined. 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 (e.g., 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. Generally, the biological activity test should provide the analysis of the desired results, such as increase or decrease in biological activity (compared to unaltered anti-CD3 antibody), different biological activity (compared to unaltered anti-CD3 antibody), receptor Affinity analysis, configuration or structure 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.

Potency, function half-life in vivo (Functional In Vivo Half-Life) And measurement of pharmacokinetic parameters :

An important aspect of the present invention is the extended biological half-life, which is obtained by constructing an anti-CD3 antibody with or without the conjugate of an anti-CD3 antibody and a water-soluble polymer portion. The rapid decrease in anti-CD3 antibody serum concentration makes it important to evaluate the biological response of treatments using conjugated and non-conjugated anti-CD3 antibodies and their variants. Preferably, the conjugated and non-conjugated anti-CD3 antibodies and variants thereof of the present invention also have an extended serum half-life after intravenous administration, so that they can be measured by, for example, an ELISA method or a preliminary screening assay. The biological half-life measurement in vivo is carried out as described in the present invention.

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

The specific activity of the anti-CD3 antibody according to the present invention can be determined by various assays known in the art. The biological activity of the anti-CD3 antibody mutein or fragments 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 :

The polypeptides or proteins of the present 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 use with a suitable pharmaceutical carrier combination. Such a composition contains, for example, a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. Formulate to suit the mode of administration. Generally, methods of administering proteins are well known in the art and can be applied to administer the polypeptides of the present invention.

According to methods well known in the art, the therapeutic composition comprising one or more polypeptides of the present invention is optionally tested in one or more appropriate in vitro and/or in vivo animal disease models to determine efficacy, tissue metabolism and estimate dosage. In particular, the activity, stability or other suitable measures (including but not limited to those modified to include one or more non-natural amino acids) of the non-natural amino acid and natural amino acid homologues in the present invention Comparison of anti-CD3 antibody and natural amino acid anti-CD3 antibody), that is, in related assays, to determine the dose.

The administration is carried out by any route normally 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 together with one or more pharmaceutically acceptable carriers. Appropriate methods for administering such polypeptides to patients are available in the context of the present invention, and although more than one route can be used to administer a particular composition, a particular route can generally provide a more direct and more effective effect than another route. reaction.

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

The polypeptide composition can 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 administration routes and appropriate formulations are generally known to those skilled in the art.

The non-natural amino acid-containing anti-CD3 antibodies, alone or in combination with other suitable components, can also be made into aerosol formulations (ie, they can be "nebulized") for administration via inhalation. The aerosol formulation can be placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration (e.g., by intra-articular (in-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antibiotics Oxidizing agents, buffers, bacteriostatic agents and solutes that make the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, the suspensions may include suspending agents, solubilizers, thickeners, stabilizers Agent and preservative. Packaged anti-CD3 antibody preparations can 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, the routes of administration that have been used for natural amino acid homolog therapy (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 pharmacologically delivering proteins) as well as the currently used formulations provide the preferred route of administration and formulation for the polypeptides of the present invention.

In the context of the present invention, the dose administered to the patient is sufficient to produce a beneficial therapeutic response in the patient over time, or includes, but is not limited to, inhibition of pathogen-caused infection or other appropriate activity, depending on the application. The dosage is determined by: the efficacy of the particular carrier or preparation, 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 dose also depends on the existence, nature, and extent of any adverse side effects that accompany the administration of a particular carrier, formulation, etc. in a particular patient.

When determining the effective amount of the carrier or preparation to be administered for the treatment or prevention of diseases (including but not limited to cancer, genetic diseases, diabetes, AIDS, etc.), the physician evaluates the circulating plasma levels and the toxicity of the preparation , Disease progression and/or (if relevant) production of antibodies against non-natural amino acid polypeptides.

The dose administered to, for example, a 70 kg patient is usually within a range equivalent to the dose of the currently used therapeutic protein, adjusted for the altered activity or serum half-life of the relevant composition. The carrier of the present invention can be supplemented by any known conventional therapies, including antibody administration, vaccine administration, cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, and biological response regulation. The application of agents, etc.

For administration, the formulation of the present invention is administered at a rate determined by the observation of any side effects of the LD-50 or ED-50 of the relevant formulation and/or various concentrations of unnatural amino acids, including but not limited to suitable for patients The rate of quality and overall health is applied. Administration can be achieved via a single dose or divided doses.

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

The human antigen-binding polypeptide of the present invention can be directly administered to a mammalian subject. Administration is by any route normally used to introduce anti-CD3 antibodies into a subject. The anti-CD3 antibody composition according to the embodiment of the present invention includes suitable for oral, rectal, topical, inhalation (including but not limited to via aerosol), buccal (including but not limited to sublingual), vagina, parenteral (including But not limited to those administered subcutaneously, intramuscularly, intracutaneously, intraarticularly, intrapleural, intraperitoneal, intracerebral, intraarterial or intravenous), topical (ie skin and mucosal surfaces, including airway surfaces) and transdermal administration, but The most appropriate route in any given situation will depend on the nature and severity of the condition being treated. Administration can be local or systemic. The formulations of the compounds can be presented in unit-dose or multi-dose sealed containers such as ampoules and vials. The anti-CD3 antibody of the present invention can be prepared as a mixture of unit dose injection form (including but not limited to solution, suspension or emulsion) and a pharmaceutically acceptable carrier. The anti-CD3 antibody of the present invention can also be administered by continuous infusion (using (including but not limited to) micropumps such as osmotic pumps), single bolus injection or sustained-release long-acting 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 can be prepared from sterile powders, granules and tablets of the kind previously described.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier depends in part on the particular composition being administered and the particular method used to administer the composition. Therefore, the present invention has a wide variety of suitable pharmaceutical composition formulations (including optional pharmaceutically acceptable carriers, excipients or stabilizers) (see, for example, "Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences), p. 17th edition, 1985).

Suitable carriers include buffers containing phosphate, borate, HEPES, citrate and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum white Protein, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, 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 nonionic surfactants, such as Tween™, Pluronics™ or PEG.

The anti-CD3 antibodies of the invention, including those linked to water-soluble polymers such as PEG, can also be administered via a sustained release system or as part of a sustained release system. Sustained release compositions include, but are not limited to, semipermeable polymer 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), polyanhydride, L-glutamic acid and γ-ethyl- Copolymers 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, polyvinyl propylene, polyvinylpyrrolidone and silicone. Sustained release compositions also include liposome-encapsulated compounds. Liposomes containing the compound can be prepared by methods known per se: DE 3,218,121; (Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034, 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; US Patent Nos. 4,485,045 and 4,544,545; and EP 102,324). All references and patents cited are incorporated into the present invention by reference.

Liposome-encapsulated anti-CD3 antibodies can be prepared by, for example, the method described in DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034, 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; US Patent Nos. 4,485,045 and 4,544,545; and EP 102,324. The composition and size of liposomes are well known or can be easily determined by those skilled in the art empirically. Some examples of liposomes are as described in, for example, the following: Park JW et al., Proc. Natl. Acad. Sci. USA 92:1327-1331, 1995; Lasic D and Papahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES, 1998; Drummond DC et al., Liposomal drug delivery systems for cancer therapy, in Teicher B (eds): 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 incorporated into the present invention by reference.

In the context of the present invention, the dose administered to the patient should be sufficient to elicit a beneficial response in the subject over time. Generally, the total pharmaceutically effective amount of the anti-CD3 antibody of the present invention administered parenterally per dose is in the range of about 0.01 μg/kg to about 100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg of the patient's body weight per day But it depends on treatment judgment. The frequency of dosing also depends on treatment judgment and may be more frequent or less frequent than commercially available anti-CD3 antibody products approved for use in humans. Generally, the bispecific antigen-binding polypeptide of the present invention can be administered by any of the above-mentioned administration routes.

The anti- CD3 Therapeutic use of antibody bioconjugates :

The anti-CD3 antibody polypeptides of the present invention can be used to treat various disorders. The anti-CD3 antibody composition disclosed in the present invention can be used to regulate the immune response. The modulation of the immune response may include stimulating, activating, increasing, enhancing or up-regulating the immune response. The modulation of the immune response may include suppression, suppression, prevention, reduction or down-regulation of the immune response. The disclosed anti-CD3 antibody-folate composition may have advantages in penetrating solid tumors, during which the natural folate ligand targets, for example, folate receptor α on tumors and folate receptor β on immunosuppressive cells. In one embodiment, the tumor is a liquid or solid tumor.

The present invention discloses a method for treating a subject's disease with the anti-CD3 conjugate or pharmaceutical composition of the present invention. In some cancers, overexpression of specific cell surface receptors can allow small molecules or drugs to selectively target cancer cells while minimizing the impact on healthy cells. For example, 2-[3-(1,3-dicarboxypropyl)-ureido]glutaric acid (DUPA) targeting specific membrane antigen of prostate cancer (PMSA) can interact with T cell surface antigen (anti-CD3) The antibody is conjugated 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}benzyl)-L-glutamic acid( Folic acid) can also be used as a biologically active molecule to bind to folate receptor (FR) antigens overexpressed on FR+ cancer cell lines.

The present invention provides a method of treating cancer by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. The cancer may be ovarian cancer, including but not limited to epithelial, mesenchymal, and germ cell tumors. Ovarian cancer can include fallopian tube cancer or primary peritoneal cancer. The cancer is 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 present invention provides a method of treating genetic diseases, AIDs or diabetes by administering to a patient a therapeutically effective amount of the anti-CD3 antibody of the present invention. In one embodiment, the anti-CD3 antibody or therapeutic agent may be a bispecific antibody comprising an anti-CD3 Fab antibody, optionally, wherein the anti-CD3 Fab antibody comprises a non-naturally encoded amine group that is position-specifically incorporated Acid, the amino acid is optionally conjugated with two folic acids and two PEGylated molecules. In another embodiment, 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 with two folic acid and two PEGylated molecules.

The present invention provides anti-CD3 antibodies for use in the treatment of diseases or disorders in cells expressing high folate receptor numbers. The anti-CD3 antibody of the present invention is used to treat cancer, including but not limited to ovarian cancer, which includes but is not limited to epithelial, mesenchymal and germ cell tumors. Ovarian cancer can include fallopian tube cancer or primary peritoneal cancer. The cancer is 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 antibody of the present invention can be used to manufacture drugs for treating diseases or disorders in cells expressing a high number of folate receptors. The anti-CD3 antibody of the present invention can be used to manufacture drugs for the treatment of cancer, including but not limited to ovarian cancer, including but not limited to epithelial, mesenchymal and germ cell tumors. Ovarian cancer can include fallopian tube cancer or primary peritoneal cancer. The cancer is 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 the treatment of 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 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 Any cancer or disease or condition related to these cancers. Carcinoma is cancer that starts in epithelial cells, which are cells that cover the surface of the body, produce hormones, and form glands. As non-limiting examples, cancer tumors include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, 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, skin or intraocular melanoma, anal area cancer, small intestine cancer, endocrine cancer Systemic cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, renal pelvis cancer, ureter cancer, endometrial cancer, cervical cancer, pituitary cancer, central nervous system (CNS) neoplasm, primary CNS lymph 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 condition. The disease or condition may be a pathogenic infection. The pathogenic infection can be a bacterial infection. The pathogenic infection may be a viral infection. The disease or condition may be an inflammatory disease. The disease or condition may be an autoimmune disease. The autoimmune disease can be diabetes. The disease or condition may be cancer. In one embodiment, the disease or condition is any disease or condition that has a highly expressed folate receptor α or β. The disease or condition may be a pathogenic infection. Biologically active molecules can interact with cell surface molecules on infected cells. Biologically active molecules can interact with molecules on bacteria, viruses, or parasites. Pathogenic infections can be caused by one or more pathogens. In some cases, the pathogen is a bacteria, fungus, virus, or protozoa. Exemplary pathogens include, but are not limited to: Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Fusiform 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 virus (for example, hepatitis A, B and C), herpes simplex Viruses (type 1 and 2), cytomegalovirus, herpes virus, HIV, influenza virus, measles virus, mumps virus, papilloma virus, parainfluenza virus, polio virus, 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 condition 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 Inflammation, erythema nodosum, giant cell arteritis (temporal arteritis), Goodpasture's syndrome, Graves' disease, Hashimoto's disease, idiopathic platelets Reduced purpura (ITP), IgA nephropathy, juvenile arthritis, diabetes, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, Lupus (SLE), mixed connective tissue Disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus, polyarteritis nodosa, type I, type II and type III autoimmune polyglandular syndrome, polymyalgia rheumatica, polymyalgia Inflammation, psoriasis, psoriatic arthritis, Reiter's syndrome, recurrent polychondrotis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, sperm and testicles Autoimmune diseases, stiff person syndrome, Takayasu's arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo and Wegener's granulation Swelling (Wegener's granulomatosis).

The disease or condition may be an inflammatory disease. Examples of inflammatory diseases include, but are not limited to, alveolitis, amyloidosis, vasculitis, ankylosing spondylitis, avascular necrosis, Baseow'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 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, fibritis/fibromyalgia, frozen shoulder, ganglion cyst, giant cell arteritis, gout, Graves' Disease, HIV Related rheumatic disease syndrome, hyperparathyroidism-related arthritis, infectious arthritis, inflammatory bowel syndrome/irritable bowel syndrome, juvenile rheumatoid arthritis, Lyme disease (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, Fidget Rheumatism, Parkinson's Disease, Plummer's Disease, Polymyalgia Rheumatica , Polymyositis, pseudogout, psoriatic arthritis, Raynaud's Phenomenon/Syndrome, Reiter's Syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, sciatica ( Lumbar radiculopathy), scleroderma, scurvy, sickle cell arthritis, Sjogren’s syndrome, spinal stenosis, lumbar spondylolisthesis, Still’s Disease, systemic lupus erythematosus, Gao’an’s (Pulseless) disease, tendinitis, tennis elbow/golf elbow, thyroid-related arthritis, trigger finger, ulcerative colitis, Wegener's granulomatosis, and Whipple's Disease.

The pharmaceutical composition containing the anti-CD3 antibody can be formulated at a strength effective for administration to human patients experiencing disorders by various means, which may be affected by anti-CD3 antibody agonists or antagonists, such as but not limited to use alone or Anti-proliferative agents, anti-inflammatory drugs or antiviral drugs are used as part of the condition or disease. The average amount of anti-CD3 antibodies can vary, especially based on the recommendations and prescriptions of qualified physicians. The exact amount of anti-CD3 antibody is a matter of priority based on factors such as the exact type of condition being treated, the condition of the patient being treated, and the other ingredients in the composition. The present invention also provides 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 with anti-CD3 antibodies, those skilled in the art can easily determine the amount to be administered.

Example:

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

Example 1:

The selection of the non-naturally encoded amino acid into the anti-CD3 antibody preferably, the location criteria: this example illustrates how to use the three-dimensional structure composed of two anti-CD3 antibody molecules, or the secondary anti-CD3 antibody , Tertiary or quaternary structure to select a preferred position in the antigen-binding polypeptide CD3 to introduce non-naturally encoded amino acids.

The following criteria are used to evaluate each position of an anti-CD3 antibody used to introduce non-naturally encoded amino acids: residue (a) should not interfere with the binding of any anti-CD3 antibody, which is based on the three-dimensional structure or secondary of the anti-CD3 antibody , Tertiary or quaternary structure, b) should not be affected by scanning mutagenesis of alanine or homologues, (c) should be exposed on the surface and exhibit minimal van der Waals or hydrogen bond interactions with surrounding residues, (d) can be on one or more exposed surfaces of the anti-CD3 antibody, (e) can be one or more positions of the anti-CD3 antibody juxtaposed with the second anti-CD3 antibody or other molecules or fragments thereof, (f) should In anti-CD3 antibody variants, it is missing or variable, (g) it will cause conservative changes after being replaced by non-naturally encoded amino acid, (h) by changing the flexibility or stiffness of the complete structure as needed, the anti-CD3 antibody itself can be adjusted Or a configuration containing one or more dimers or multimers of anti-CD3 antibodies, (i) may be present in a highly flexible region or a structurally rigid region, and (j) may or may not be present in the complementarity determining region ( CDR). In addition, the Cx program (Pintar et al., Bioinformatics, 18, page 980) was used to perform further calculations on the anti-CD3 antibody molecule to evaluate the protruding degree of each protein atom. As a result, in one embodiment, the non-naturally 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 Escherichia coli: the introduction and translation system containing orthogonal tRNA (O-tRNA) and orthogonal amino acid tRNA synthetase (O-RS) was used to express the Non-naturally encoded amino acid anti-CD3 antibody. O-RS preferentially utilizes non-naturally encoded amino acids to aminate O-tRNA. 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 Escherichia coli with a plastid containing a modified anti-CD3 antibody gene and an orthogonal amine-based tRNA synthetase/tRNA pair (specific for the desired non-naturally encoded amino acid), allowing the non-naturally encoded amine to be transformed The base acid position is specifically incorporated into the anti-CD3 antibody. Transformed Escherichia coli grown at 37°C in a medium containing 0.01-100 mM of specific non-naturally encoded amino acids expresses modified anti-CD3 antibodies with high fidelity and efficiency. Anti-CD3 antibodies containing non-naturally encoded amino acids are produced as soluble proteins in the periplasm of E. coli host cells. The method of purifying anti-CD3 antibodies is well-known in the art, and has been confirmed by SDS-PAGE, Western blot analysis, or electrospray ionization ion trap mass spectrometry.

Expression/inhibition: Inhibition of acetanilide (pAF) with a non-naturally encoded amino acid: The suppression of amber mutations in E. coli is achieved using standard protocols known in the art. In short, in order to perform periplasmic suppression of antibody fragments in Escherichia coli (Fab), the expression vector constructs were used to encode orthogonal tRNA synthetase (for example, orthogonal tyrosine from M. jannaschii). The plastids of amine-tRNA synthetase (MjTyrRS) were transformed into E. coli host cells. The overnight bacterial culture was diluted 1:100 into a shake flask containing LB medium (Luria-Bertani) or Superbroth, and grown at 37°C to an OD of about 0.8. The Fab expression is induced by adding p-acetylphenylalanine (pAF) to a final concentration of 4 mM and the suppression of amber codons is achieved at the same time. The culture was incubated overnight at 25°C (incubated overnight).

Use non-naturally encoded amino acid derivatives for inhibition: use non-naturally encoded amino acid derivatives (such as pAF (aa 9.2)) to inhibit amber mutations. The implementation is similar to the above, the difference is that the use of p-amino groups Acid-specific orthogonal synthetase (for example, Tyramine-tRNA synthetase (MjTyrRS) from Methanococcus jannaschii). For example, inhibition can be achieved by adding aa9.2 (4 mM) at the time of induction.

The cells were collected by centrifugation and resuspended in periplasmic release buffer (50 mM NaPO4, 20% sucrose, 1 mM EDTA, pH 8.0) supplemented with 100 ug/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 help of its His tag, the beads were washed thoroughly with binding buffer and the bound fragments were eluted from the beads with 0.5 M imidazole . The purified fragment was dialyzed in storage buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 5% sucrose, pH 7.8). For small-scale analysis of Fab fragments expressed in the periplasm, 15 ml of E. coli cultures were collected by centrifugation and resuspended in 1 ml of lysis buffer supplemented with 10 ug/ml DNA enzyme (B-PER, Pierce Biotechnology; Rockford, IL). The mixture was incubated at 37ºC for 30 minutes, diluted to 1X in protein loading buffer (Invitrogen; Carlsbad, CA) and analyzed by SDS-PAGE.

Example 3:

Design and construction of humanized anti-CD3 gene in pFUSE vector-Humanized mouse monoclonal anti-CD3 antibody SP34 (Harvard BIDMC). According to computer analogy analysis and design, in addition to the mouse CDR sequence in the selected human framework scaffold, 3 variable heavy chains (vH1.0, vH1.1 and vH1.2) 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 (back mutations) and their Kabat numbers, which are retained in the 3 variable heavy chains (vH) and 3 variable of the 9 humanized anti-CD3 Fab mentioned in Figure 1. Light chain (vL). The amino acid residues restored to the human framework sequence are shown in bold.

surface 2 - List of mouse framework residues

As shown in Table 2, there are 5 mouse back mutations in the variable light chain (V36, G46, G49, G57, and V58) and 4 mouse back mutations in the variable heavy chain (N30, A49, I77, and V93). ). The 6 short synthetic variable genes shown in Table 2 were cloned into Invivogen's heavy chain (HC) and light chain (LC) expression vectors (respectively pFUSE-CHIg-HG1 and pFUSE-CLig- In hk), a plastid expressing humanized anti-CD3 SP34 antibody is generated. As shown in Figure 1 and Table 2, the variable light chain vL1.0, in which all 5 mouse backmutations are converted to human residues, loses binding to any variable heavy chain (vH) combination.

Example 4:

The expression of humanized anti-CD3 antibody in HEK293 transient system: By combining each plastid containing vH gene with each plastid containing vL gene (co-transfection), the human described in the previous example The SP34 antibody was transiently expressed into HEK293 cells. Determine the protein concentration in the cell culture medium from each co-transfection experiment and use it directly in the PBMC-based CD3 binding assay without further purification. The controls used were the chimeric SP34 construct (positive control) and the unrelated PSMA antibody construct (negative control). Humanized anti-CD3 antibodies were also expressed in HEK293 cells, which did not incorporate unnatural amino acids, and had or did not have the HC-DKTHT extension, and were characterized (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 the humanized anti-CD3 heavy chain (vH+CH1) and light chain (vL+CL) sequences, which are used in different combinations to produce the Fab 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 the binding of CD3 to activated human and cynomolgus PBMC: In order to distinguish the generated humanized anti-CD3 antibodies, activated human PBMC (n=2) was used to test the binding to human CD3. The activation of PBMC and the subsequent fluorescence-based CD3 binding assay were followed as previously described (see, for example, Angew Chem Int Ed Engl, 52(46): 12101-12104, 2013). As shown in Figure 1, the three antibodies with vL1.0 light chain lost binding to human CD3 with any vH heavy chain combination. Activated human and cynomolgus monkey PBMC were used to titrate the binding of the remaining 6 humanized antibodies to human and cynomolgus monkey CD3. As shown in Figures 2A-2F, all 6 humanized anti-CD3 antibodies (engineered by the combination of 3 vH heavy chains and 2 vL light chains shown in Table 3) have retained the compatibility with both human and cynomolgus CD3 Quite a combination. Six kinds of humanized anti-CD3 antibodies were obtained as follows: by 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)) the combination produces Fab1 and Fab2, respectively 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) ); (SEQ. ID. NO: 2)) and ((vH1.0 + CH1); (SEQ. ID. NO: 3)) to produce Fab4, Fab5, and Fab6 wild-types, respectively.

Since the data showed that there was no significant difference in binding two CD3 substrates between the 6 humanized antibodies, Fab1 (vH1.2+vL1.2 combination) was selected to further prove the key aspects of the present invention. For example, Fab1 is used for expression in E. coli to be further optimized by the previously described orthogonal amber suppression system using proprietary unnatural amino acid (UAA) incorporation technology (see e.g. WO2017/079272, WO2012/166560 And WO2013/192360). Table 4 shows the list of mouse framework back mutations necessary to retain binding activity in these 6 Fabs, and Table 4 provides the mouse frameworks mentioned in Figure 2A-2F that are retained in the 6 humanized anti-CD3 Fabs. List of residues (back mutations) and their Kabat numbers. The amino acid residues restored to the human framework sequence are shown in bold. The vL1.2 and vH1.2 variable chain combination (underlined) producing Fab1 was selected as the leader for further modification.

Table 4-Mouse framework residues retained in 6 humanized anti-CD3 Fabs

Example 6:

Clone the synthesized Fab gene into an E. coli expression vector: Use the Gibson Assembly cloning kit (New England Biolabs) to clone the synthesized Fab gene into a proprietary standard E. coli expression vector. After sequence verification of each expression plastid, each plastid was transformed into a standard E. coli production host W3110B60 strain, and the isolated single colony of each plastid was purified to make a glycerol vial. The glycerol vials were used as production clones for E. coli fermentation of these Fab molecules. The amino acid sequences of the 4 heavy chains and 3 light chains used to engineer these Fabs are shown as SEQ. ID. NO: 10 to 20 in Table 5.

Table 5-Humanized anti-CD3 Fab1 (vH1.2+CH1, vL1.2+CL) amino acid sequences of heavy chain and light chain sequences produced in E. coli, where the non-natural amino acid pAF is incorporated into the position Mark it with an underline.

三條輕鏈(LL157pAF、LK172pAF和LS205pAF) pAF變體(SEQ. ID. NO: 18至20)以及3種雙pAF變體((HK129pAF+LL157pAF)；(SEQ. ID. NO: 16和18))；((HK129pAF+LK172pAF)；(SEQ. ID. NO: 16和19))和((HK129pAF+LS205pAF) (SEQ. ID. NO: 16和20))被工程改造。SEQ. ID. NO: 10至13代表不具有5-aa重鏈C末端延伸(DKTHT)的重鏈pAF變體。

Three light chains (LL157pAF, LK172pAF and LS205pAF) pAF variants (SEQ. ID. NO: 18 to 20) and three 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 5-aa heavy chain C-terminal extension (DKTHT).

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

By aseptically transferring the contents of the inoculum, the production fermentor was inoculated at a target OD600 of 0.0004. After inoculation, the culture was sampled at appropriate intervals to determine OD600. Monitor temperature, pH, and dissolved oxygen and control them at 37°C, 7.0, and >30% specified set points, respectively. The pH is controlled by adding ammonium hydroxide solution or sulfuric acid. The dissolved oxygen is controlled by changing the stirring speed and by increasing the oxygen composition in the sprayed air/oxygen mixture. Anti-foaming agent is added during fermentation to control foaming.

When the cell density reaches OD600> 25, add a batch of feed medium. When the cell density reached OD600> 50, the feed medium was added at a constant flow rate of 0.094 mL/L starting volume/min for 32 hours, until the fermentation was finished and it decreased to 0.052 mL/L starting volume/min. Immediately after the feed is started, a specified amount of non-naturally encoded amino acid (for example, pAF) solution is added aseptically, so that the non-natural amino acid is incorporated into the protein amino acid sequence. At the same time, the temperature was changed from 37°C used during growth to 27°C for production. Production is controlled by the phoA activator and starts when the phosphate level in the medium is exhausted. The collection started about 48 hours after induction.

Example 7:

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

After lysis, the WCL was centrifuged at 15,900 x g for 30 minutes at 4°C and filtered (0.8/0.2 microns) to remove precipitated proteins and cell debris. Capto S cation exchange chromatography was then used to capture the anti-CD3 Fab from the E. coli WCL supernatant. After the Capto S column, Butyl HP Hydrophobic Interaction Chromatography (HIC) is used as a polishing column to separate the intact anti-CD3 Fab from the product-related impurities present in the Capto S elution pool. Then the Butyl HP elution cell containing intact anti-CD3 Fab was buffer exchanged to 50 mM acetate, 5% trehalose, pH 4.0 and concentrated to prepare for conjugation with folic acid or PEG-folic acid. This step was carried out at 4°C and used an Amicon Ultracel 10K regenerated cellulose (15 mL) centrifugal device.

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

CD3 Fab folic acid-5KPEG, CD3 Fab folic acid-10KPEG, CD3 Fab folic acid-20KPEG, CD3 Fab folic acid-(5K)2PEG, CD3 Fab folic acid-(10K)2PEG compounds were produced. At 28°C, the desired folate-PEG (5K, 10K, 20K, 5K2 or 10K2 PEG) was added to the CD3 Fab buffer solution (50 mM acetate, 5% trehalose, pH 4). After one hour, the mixture was purified by cation exchange chromatography (Toyo SP 5PW) and buffered with 50 mM histidine, 100 mM NaCl, 5% trehalose, pH 6.0 by using a centrifugal filter (c/o 10K) Liquid preparation to obtain the desired CD3 Fab-folate-pegylated composition. The structure, chemistry and conjugation of CD3 Fab folate conjugate are described in the present invention and shown in Figures 12A-12D.

In addition, a CD3 Fab folate-pegylated C-terminal conjugate was produced. EDTA (6.7 uL, 0.5 M, pH 8) and DTT (0.4 mg) were added to CD3 folic acid (8.0 mg) in PBS (2.0 mL), and the solution was incubated at 37°C for 30 minutes. Purify the mixture through a desalting column using 5 mmol EDTA in PBS. At room temperature, various concentrations of Mal-PEG (4.2 mg 5K, 8.2 mg 10K, or 16 mg 20K) were added to the mixture. After 2 hours, the mixture was purified by Toyo SP 5PW cation exchange chromatography to obtain CD3 Fab folic acid-(PEG5K)2 C-terminal conjugate, CD3 Fab folic acid-(PEG10K)2 C-terminal conjugate and CD3 Fab folic acid-(PEG20K) ) 2 C-terminal conjugate. The structure, chemistry and conjugation of the C-terminal PEG conjugate are described in the present invention and shown in Figures 12E-12F.

Example 8:

In vitro binding and killing assay: The purified humanized anti-CD3 Fab conjugated with folic acid at different heavy chain positions (HA114, HS115, HK129, HT160) was initially tested for binding to both human and cynomolgus CD3. The corresponding unconjugated protein with pAF at the position and the wild-type protein served as controls. Table 6 depicts the EC50 of the modified anti-CD3 Fab1 protein purified from E. coli cells to activate human and cynomolgus PBMC. As shown in Table 6, neither pAF nor conjugated folic acid at different positions significantly interfered with its CD3 binding.

表6. 修飾的抗CD3 Fab1蛋白的結合

Table 6. Binding of modified anti-CD3 Fab1 protein

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

As shown in Table 7, there is no significant difference in killing EC50 between the folic acid conjugate positions. However, compared with no SFA, when 50 nM SFA is present, the cytotoxic EC50 is observed to be about 15-60 times lower due to the competitive inhibitory effect of free SFA. In addition, for each variant, the EC50 of cynomolgus monkeys is about 10 times higher than that of humans.

表7 - 修飾的抗CD3 Fab1蛋白對SKOV-3細胞的細胞毒性

Table 7-Cytotoxicity of modified anti-CD3 Fab1 protein to SKOV-3 cells

Example 9:

Cytotoxicity of anti-CD3-folate variants on multiple FOLRα tumor cell lines with different levels of folate receptor (FRα): In vitro cytotoxicity assays using 6 different cell lines with different levels of FRα overexpression on the cell surface , Tested the activity of three different anti-CD3 Fab-folate conjugates at the positions of 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. The cells were co-cultured with activated human peripheral blood mononuclear cells (PBMC; target: effector cell ratio 1:10) and treated with various concentrations of anti-CD3 Fab-folate in the presence of 50 nM SFA. The cytotoxicity was quantified by measuring the level of LDH released from lysed cells and using CellTiter-Glo and FACS-based toxicity assays.

Table 8-Cytotoxicity of anti-CD3-folate variants on multiple FRα tumor cell lines

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

Example 10

This example shows the effect of folic acid-PEG conjugation at a single position or the addition of a second folic acid (difolate) 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 of conjugating a 5K or 20K linear PEG molecule with folic acid using the bifunctional linker as described in the present invention on the binding and Table 10 shows the cytotoxicity. Depending on the size of the 5K or 20K PEG, a decrease in knot fitness (1.7 to 8.4 times) was observed for both activated human and cynomolgus PBMC. However, for both human and cynomolgus PBMC, the cytotoxic activity when using 20K PEG is greatly reduced compared to 5K PEG (3.7 to 5.6 times vs. 50 to 58 times). According to this experiment, 5K PEG was used instead of 20K PEG to extend the half-life.

表9 – CD3結合

Table 9-CD3 binding

表10 - 細胞毒性

Table 10-Cytotoxicity

The effect of dual folic acid (difolate) conjugate: Table 11 shows the effect of the second folic acid conjugated heavy chain HK129 position on the two light chain positions (LL157 and LK172) on the in vitro binding, and Table 12 shows the cytotoxicity . Sure enough, compared with the HK129 monofolate molecule, a moderate increase in the binding affinity and cytotoxic activity of the difolate variant was observed. In the presence or absence of 50 nM SFA, a modest increase in the cytotoxicity of difolate molecules relative to monofolate molecules is also evident. According to this experiment, the LL157 position is better than the LK172 position for combination with the HK129 position for future research.

表11 - CD3結合

Table 11-CD3 binding

表12 – 細胞毒性

Table 12-Cytotoxicity

Summary of binding affinity and in vitro cytotoxicity: Table 13 summarizes the effects of pegylation and second folate conjugation on the binding and cytotoxic activity of various anti-CD3 Fabs. A moderate decrease in binding affinity to both CD3 and FRα will result in a significant decrease in potency, and is related to the size of PEG. Although the CD3 binding affinity has been reduced, the conjugation of difolate molecules increases the affinity for FRα, thereby increasing the cytotoxicity. Although the affinity for the two targets is reduced due to PEG conjugation, the in vitro killing EC50 of all CD3-Fab-folate molecules still maintains a high potency in the range of 37 to 335 pM.

表13 – 各種聚乙二醇化抗CD3 Fab的結合和細胞毒活性。

Table 13-Binding and cytotoxic activity of various PEGylated anti-CD3 Fabs.

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 E:T ratio on SKOV-3 The effect of cell cytotoxicity, in the presence of 50 nM SFA at 10:1, 5:1, 1:1, 1:5 and 1:10 cytotoxicity assay of 3 kinds of anti-CD3 Fab1 molecules . As expected and as shown in Table 14, the E:T ratio is correlated with killing potency in vitro. Under E:T=1:1, it was observed that the EC50 of difolate molecules increased by 2 times, while the EC50 of 5KPEG-folate molecules decreased by 8 times. Within an E:T ratio of 10:1 to 1:10, all three molecules remain highly effective, with EC50 ranging from 1.69 to 175 pM.

表14 – 效應細胞:靶細胞(E:T)比率對體外細胞毒性的影響

Table 14-Effect of effector cell: target cell (E:T) ratio on in vitro cytotoxicity

Example 12:

Pegylation of anti-CD3 Fab1 molecules to extend plasma half-life: The pharmacokinetic properties of anti-CD3 Fab1-folate conjugates in the presence and absence of PEG conjugates in rats were studied. Male Sprague Dawley rats (approximately 7 weeks old) were used. On the day of administration, the body weight of each animal was measured. 1 mg of non-conjugated and conjugated anti-CD3 antibody samples per kg body weight were each injected intravenously into the tail veins of three rats. At different time points after the injection, 500 μl of blood was drawn from each rat under CO2 anesthesia. The blood samples were stored at room temperature for 1.5 hours, and then the serum was separated by centrifugation (4°C, 18000 x g, 5 minutes). The serum samples were stored at -80°C until the day of analysis. After the samples were thawed on ice, the amount of active anti-CD3 antibodies in the serum samples was quantified by the in vitro activity determination of anti-CD3 antibodies.

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

表15 – 各種Fab1分子在大鼠中的藥物代謝動力學

Table 15-Pharmacokinetics of various Fab1 molecules in rats

Example 13

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

De-convolution of mouse back mutations and germline mutations (De-convolution): Mouse framework residues (reverted mutations) of both vH and vL sequences obtained during the humanization of anti-CD3 antibodies are passed one at a time The ground is restored to human germline residues to examine its effect on antigen binding (deconvolution). For the vH sequence, the 4 mouse framework residues (back mutations) at Kabat positions 30, 49, 77 and 93 are expected to play a key role in antigen binding (Tables 2-3 and 16-17). Similarly, for the vL sequence, the five mouse framework residues (back mutations) at Kabat positions 36, 46, 49, 57 and 58 are also expected to play a key role in antigen binding.

表16 - 抗CD3 Fab1輕鏈(LC)回復突變和種系突變位置

Table 16-Anti-CD3 Fab1 light chain (LC) back mutations and germline mutation positions

表17 - 抗CD3 Fab1重鏈(HC)回復突變和種系突變 (germ-line mutation)位置

Table 17-Locations of anti-CD3 Fab1 heavy chain (HC) back mutations and germ-line mutations

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) plastids were generated (Table 16- The first round plastids in 17). These vH and vL plastids were used in the HEK293 cell co-transfection experiment together with the original vH and vL plastids described in the above examples (round 0 plastids in Table 16-17). A total of 35 transient transfections were performed in the first round of screening, and as described below and in the above examples, the cell culture supernatant was directly used to screen for binding.

According to the screening results of the first round, three additional vH plastids (vH2.4 to 2.6) were generated (the second round plastids in Table 16-17) to evaluate the cumulative effect of each back mutation. In a similar way, a new vL plastid (vL2.5) was generated by combining two mouse backmutations. In addition, mouse germline mutations present in the vH and vL CDRs conferred reduced binding in this round were analyzed. To this end, by changing the amino acid of Kabat position N35 in HC-CDR1 and Y52c in HC-CDR2, three new vH plastids (vH3.1 to 3.3) were generated (the second round plasmid in Table 16-17). body). In a similar manner, vL plastids (vL3.1) were generated by changing the amino acid of Kabat position K53 in LC-CDR2 (the second round plastids in Table 16-17). By selectively combining various vL/vH plastid pairs, a second round of screening experiments involving a total of 43 transfections was carried out.

Screening for CD3 binding between humans and cynomolgus monkeys: In the screening stage, the HEK293 culture supernatant was directly used to test the 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 with both human and cynomolgus CD3. For each clone, an 11-point binding titration curve was generated for both human (Figure 4A) and Cynomolgus CD3 (Figure 4B). According to the data, Fab 7 to 10 were selected as low-affinity candidates for further evaluation (Figure 4A-4B). Similarly, 43 clones were screened in the second round of experiments. After 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 to the E. coli expression system for use in non-natural amino acids ( The incorporation of UAA) and further testing as described in the examples below. Figure 6A shows the Fab-folate conjugate of the same wild-type Fab as shown in Figure 4A. The Fab-folate conjugates representative of wild-type Fab obtained from the second round of screening are depicted in Figure 6B. Table 18 shows a list of low-affinity mutations and their HC, LC and Fab ID. As shown, both mouse framework back mutations and germline CDR mutations played an important role in conferring reduced binding to both human and cynomolgus CD3. In the variable light chain (vL), 5 mouse recovery residues at Kabat positions V36, G46, G49, G57, and V58 in LC-CDR2 and one mouse germline residue at Kabat position K53 are involved in reduced binding . In the variable heavy chain (vH), the four mouse recovery residues at Kabat positions N30, A49, I77 and V93 in HC-CDR1 and two mice at Kabat position N35 and HC-CDR2 at position Y52c Germline residues are involved in reduced binding.

表18 - 低親和力人源化抗CD3 Fab變體中的突變列表

Table 18-List of mutations in low-affinity humanized anti-CD3 Fab variants

表19 – 在HEK293細胞中篩選低親和力Fab變體中所用的人源化抗CD3重鏈(vH+CH1)、(vH+CH1-DKTHT)和輕鏈(vL+CL)的胺基酸序列

Table 19-Humanized anti-CD3 heavy chain (vH+CH1), (vH+CH1-DKTHT) and light chain (vL+CL) amino acid sequences used in screening low-affinity Fab variants in HEK293 cells

SEQ. ID. NO: 21 to 29 represent the amino acid sequence of the heavy chain with 5-aa C-terminal extension (DKTHT) used in the low-affinity variant Fab screening. SEQ. ID. NO: 30 and 38 represent these humanized heavy chain amino acid sequences without 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 produced from E. coli cells using the heavy chain HK129 amber mutation.

Cloning into E. coli expression vector: Design synthetic gene for all low-affinity variants disclosed in Table 20, SEQ. ID NO: 40-62 has STII-LC-spacer-STII-HC expression cassette structure, in the heavy chain The amber TAG stop codon was inserted at HK129 position and cloned into a proprietary E. coli expression vector, as described in the above example. 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 5-aa heavy chain C-terminal extension (DKTHT) used in the test. SEQ. ID. NO: 49 and 57 represent these humanized heavy chain HK129 pAF variants without 5-aa heavy chain C-terminal extension (DKTHT). SEQ. ID. NO: 58-62 represent the light chain sequence used in combination with the HK129pAF-DKTHT variant of SEQ. ID. NO: 40 to 48, which was expressed in E. coli and further characterized.

表20 – 在大腸桿菌中表達的低親和力抗CD3 Fab-HK129pAF重鏈和輕鏈序列的胺基酸序列。還公開了下表中的所有序列，其中pAF由任何其它非天然胺基酸代替。

Table 20-The amino acid sequences of the heavy and light chain sequences of the low-affinity anti-CD3 Fab-HK129pAF expressed in E. coli. All sequences in the table below are also disclosed, where pAF is replaced by any other non-natural amino acid.

Fermentation, purification and folic acid conjugation: E. coli fermentation, purification, folic acid conjugation and post-conjugation purification were carried out as described in the above examples.

Binding of folate-conjugated Fabs to human and cyno CD3: The binding of low-affinity variants of humanized anti-CD3 Fab produced by E. coli and conjugated with folate was performed as described in the above examples. Figures 6A-6B show the binding affinities of two subsets of low-affinity Fab variants of anti-CD3 Fab-HK129-folate and the positive control Fab1 to human CD3. Among the tested low-affinity Fabs, even at the highest tested concentration (1000 nM), 10 failed to significantly bind to human CD3. Compared to the 2.31 nM of the control Fab1, the binding EC50 of the remaining 4 Fabs (Fab 7 to 10; described in Table 18) ranged from 23.4 nM to 83.6 nM. Fab 21 shows weak binding activity and will not saturate even at a concentration of 1000 nM. As shown in Figures 7A-7B, very similar binding profiles were observed for cynomolgus CD3 binding.

The cytotoxicity assay of folate-conjugated Fab was performed with human and cynomolgus PBMC: The cytotoxicity of low-affinity Fab was performed as described in the above example. Figure 8 shows the cytotoxicity data of activated human PBMC using SKOV-3 cells. As shown, all 4 Fabs (Figures 6A-6B) with a 10- to 36-fold reduced binding affinity to human CD3 showed comparable killing activity compared to the control Fab1 (Figure 8). Three Fabs (Fab 11, 19 and 20) did not show any cytotoxic activity (data not shown). All the other 8 Fabs showed considerable killing activity, but they could not bind even at a concentration of 1000 nM. A very similar cytotoxicity profile was observed using activated cynomolgus monkey PBMC (Figure 9).

Example 15:

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

T cell activation/cytokine release assay: Separate and purify human T cells and T cells from the same volume of whole blood by EasySep Human T Cell Enrichment Kit and EasySep Human CD3 Positive Selection Kit (STEMCELL Technologies Inc) Human PBMC. The purity of the isolated T cells and helper cells was confirmed by flow cytometry. In order to selectively monitor T cell activation in the presence of helper cells, the purified T cells were labeled with the Cellvue Lavender Cell Labeling Kit (eBioscience) according to the manufacturer's protocol before mixing with T cell depleted human PBMC. In the presence of the anti-CD3 Fab variant, the resulting reconstituted PBMC is cultured with target cells. After 48 hours, the 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 the culture supernatant was measured by an enzyme-linked immunosorbent assay (ELISA) kit (R&D System).

As shown in Figures 10A-10B, the significant T cell activation (through CD25 and CD 69 T cell markers) achieved in the presence of SKOV-3 cells strongly depends on the dose of various low-affinity anti-CD3 Fab-HK129-folate molecules . For the cytokine release of IFNγ in the presence and absence of SKOV-3 tumor cells (Figure 11A-11B) and TNFα in the presence and absence of SKOV-3 tumor cells (Figure 11C-11D), respectively Measurement, a similar correlation was observed.

Summary of low-affinity variant characterization: Table 21 summarizes the binding and cytotoxicity data of 12 low-affinity anti-CD3 Fab variants with HK129-folate modification (human and cynomolgus CD3), and T cell activation (CD25 and CD69 markers) ) And cytokine release (IFNγ and TNFα) determination. 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.

According to the data set, three low-affinity variants Fab9, Fab10, Fab21 and 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 regard to cytotoxicity and the production of two 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 series of anti-CD3 Fab concentrations in which the release of cytokine will not cause major safety issues, but the killing efficacy and T cell activation will not be impaired. This method of fine-tuning the affinity of anti-CD3 antibodies may allow the decoupling of these two relative events and achieve a better safety profile without compromising efficacy.

表22 – 具有HK129-葉酸修飾的3個所選低親和力變體的表徵

Table 22-Characterization of 3 selected low-affinity variants with HK129-folate modification

Example 16 :

In-silico immunogenicity analysis of anti-CD3 Fab1, 9 and 10: In order to evaluate potential immunogenicity, based on Lonza's Epibase platform, use the "HLA Class II-Global Version 4.0" setting (Lonza, UK), scanned the amino acid sequences of anti-CD3 Fab1, Fab9, and Fab10 by computer analogy, putative human leukocyte antigen (HLA) class II restricted epitopes (also known as T helper (Th) cell epitopes) The presence. The HLA binding specificity of all possible 10-mer peptides derived from the target sequence was analyzed. At the allotype level, 43 DRB1, 8 DRB3/4/5, 22 DQ and 12 DP (ie a total of 85 HLA II allotypes) were analyzed. The peptides corresponding to the self-peptides are treated separately as "germline filtering" 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 antigens, the observed activation/proliferation of Th cells is usually 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 score of each of these 3 Fabs in the global population is comparable to humanized therapeutic antibodies. Among the 3 Fabs, Fab9 has the lowest immunogenicity.

表23. 對應於DRB1、DRB3/4/5、DQ和DP基因的HLA結合物(表位元數目)。

Table 23. HLA binders (number of epitopes) corresponding to DRB1, DRB3/4/5, DQ and DP genes.

Table 24. DRB1 risk scores of 3 Fabs in the global population.

Example 17 :

The design and synthesis of bi-functional PEG-Folate linkers are 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 - Trifluoroacetyl pteroic acid (2) . Under nitrogen, 10 ml of trifluoroacetic anhydride was introduced dropwise to 1.0 g of pteroic acid (1) in a round bottom flask for 10 minutes. The reaction mixture was stirred at room temperature for 24 hours, protected from light. The dark brown solution was filtered through a pad of Celite and evaporated. The resulting viscous brown oil was wet-milled with ether, and the separated precipitate was collected by filtration, washed with ether, and dried under vacuum overnight to obtain a crude intermediate in the form of light brown powder. The crude acylated material was resuspended in anhydrous THF and treated with ice. The resulting mixture was stirred at room temperature for 10 hours; during this time a light brown precipitate separated. The reaction mixture was diluted with diethyl ether, and the precipitate was collected by filtration, washed with diethyl ether and dried under vacuum overnight to obtain N10-trifluoroacetylpteroic acid (2) (1.45 g crude substance) as a light brown powder. Further purification is used in the next reaction.

N ¹⁰ - butterfly trifluoromethyl acetylsalicylic acid OSu ester (3). At room temperature, a solution of N10-trifluoroacetylpteroic acid (2) in anhydrous DMSO was treated with N-hydroxysuccinimide (0.43 g), followed by EDCI-HCl (2.04 g) in aliquots . 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), MS (ESI) m/z 506 (M+H)+ as a brown powder.

Fmoc-Glu-OtBu-Lys(Boc)-OtBu (7 ). At room temperature, 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 . 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 obtain crude Fmoc-Glu(OSu)-OtBu (5) (5.3 g) as a white foam. 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 to be complete by HPLC analysis, and the solvent was removed under vacuum. The residue was redissolved in ethyl acetate (100 mL), washed with 10% aqueous citric acid (50 mL), water (50 mL), and brine (50 mL), and dried over sodium sulfate. After the solvent was removed in vacuo, the residue was wet 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 to be complete by HPLC analysis. All solvents were removed in vacuo, and the residue was purified by silica gel column chromatography eluting first with dichloromethane and then with 5-10% MeOH in dichloromethane to give H-Glu-OtBu-Lys(Boc )-OtBu (8).

Compound 9: A solution of amine 8 in anhydrous DMF was treated with N10-trifluoroacetoxypteric acid OSu ester (3) as a whole. The resulting mixture was stirred at room temperature for 24 hours while monitoring its completion by HPLC analysis. The reaction mixture was diluted with ethyl acetate and filtered through a pad of silica gel eluted with a 10% methanol in ethyl acetate solution. The combined filtrates were evaporated to dryness, redissolved in ethyl acetate, and washed successively with 10% aqueous citric acid, water, saturated NaHCO3 and brine. The extract was dried over sodium sulfate, evaporated and dried under vacuum overnight to give crude 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 the overall deprotection was judged to be complete by HPLC analysis. All solvents were removed under vacuum, and the remaining brown oil was wet milled with ether and sonicated briefly. The separated light-colored precipitate was collected by filtration, washed thoroughly with ether and dried in vacuum for one day to obtain product 10 in the form of 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 11 was observed to be completely consumed by HPLC analysis. The reaction mixture was diluted with pH 5 acetate buffer (0.5M) and 1 mL of acetonitrile, and purified by C18 reversed-phase HPLC using a gradient of 20-90% acetonitrile/0.05% TFA as the eluent, and it was lyophilized to give a white Compound 12 in solid form.

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

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

Compound 16: At room temperature, add small pieces of sodium to the solution of tetraethylene glycol 14 in anhydrous THF, 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 under vacuum and resuspended in brine, followed by extraction with ethyl acetate. The combined organic phase was washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give compound 16 as a clear light yellow oil.

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

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

Compound 19: The acetate 18 (t -Butyl ester 18) 1 with TFA and DCM at room temperature: 1 mixture treated. After 3 hours, the solvent was removed in vacuo, the residue was dissolved in dichloromethane, washed thoroughly with brine and dried over sodium sulfate. After the solvent was removed under vacuum, the crude carboxylic acid 19 was obtained in the form of a light yellow clear oil.

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

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

Compound 21: Boc-Lys-OH 20 At 23° C., to a solution of OSu ester 11 (1.55 g) and Boc-Lys-OH 20 (0.72 g) in DCM (50 ml) was added DIEA (1.03 mL). After 10 minutes, LCMS showed that the reaction was complete. The mixture was washed with 1N HCl (50 ml), saturated sodium bicarbonate (50 ml) and brine (50 ml). The organic layer was dried with MgSO4. The solvent was removed to obtain 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 treatment 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 gel column chromatography using a 0-10% methanol/DCM gradient as the 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). The organic layer was dried with 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 treatment with DCC at room temperature. After 4 hours, the mixture was filtered to remove DCU and concentrated in vacuo. The residue was purified by silica gel column chromatography using a 0-6% methanol/DCM gradient as eluent to give 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 material (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 with a gradient of 5% to 60% water/90% ACN 0.05% TFA for 20 minutes. The fractions containing the product were combined and evaporated in vacuo to give compound 26 as a brown solid; MS (ESI) m/z 1535 (M+H)+.

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

Compound 29A: To a solution of compound 27 (0.054 g) and compound 28A (PEG5K-C5-NHS, 0.17 g) in DMF (2 ml) was added DIEA (0.034 mL) at 23°C. After stirring for 5 hours, the mixture was dropped on 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 obtain compound 29A as a crude white solid.

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

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

Compound 30B: To a solution of compound 29B (0.47 g) in water (6 ml) at 23°C was added hydrazine·H2O (0.080 mL). After stirring for 6 hours, the mixture was purified by preparative LC using a C18 column using a 20% to 100% ACN and 0.05% TFA water gradient for 20 minutes. The fractions containing the product are 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) was added DIEA (0.040 mL) at 23°C. After stirring for 16 hours, the mixture was dropped on 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 minutes, 4000 rpm). The solvent was decanted, and the white precipitate was dried under high vacuum overnight to obtain compound 29C as a crude white solid.

Compound 30C: To a solution of compound 29C (0.67 g,> 0.031 mmol) in water (8 ml) was added hydrazine·H2O (0.060 mL) at 23°C. After stirring for 6 hours, the mixture was purified by preparative LC using a C18 column using a 20% to 100% ACN and 0.05% TFA water gradient for 20 minutes. The fractions containing the product are 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 on 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 obtain compound 29D as a crude white solid.

Compound 30D: To a solution of compound 29D (0.45 g,> 0.021 mmol) in water (8 ml) was added hydrazine·H2O (0.080 mL) at 23°C. After stirring for 48 hours, the mixture was purified by preparative LC using a C18 column using a 20% to 100% ACN and 0.05% TFA water gradient for 20 minutes. The fractions containing the product are 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 with water to 1 mL, and purified by a desalting column. The collected fraction was lyophilized to obtain compound 28E as a white solid.

Compound 30E: To a solution of compound 28E (0.04 g), DMTMMT (0.003 g) and DIEA (0.005 mL) in DMF (2 mL) was added compound 27 (0.009 g) at 23°C. After stirring for 1 hour, LCMS showed that the reaction was complete. To this mixture (crude compound 29E), hydrazine·H2O (2 ul) was added in situ. After stirring for 1 hour, the mixture was purified by preparative LC using a C18 column with a water gradient of 20% to 100% ACN and 0.05% TFA for 20 minutes. The fractions containing the product are combined and evaporated. The residue was dissolved in water (10 ml) and lyophilized to give compound 30E as a pale yellow solid.

Compound 31: Add 20K-PEG-amine (0.31 g) and 2-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) ethyl To a solution of 2,5-dioxopyrrolidin-1-yl acetate (0.003 g) in DMF (1.0 ml) was added DIEA (0.006 mL). After 30 minutes, the mixture was purified with 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 in this invention.

Example 18 :

This example illustrates the construction, expression and purification of humanized anti-CD3 Fab lead molecules containing double amber. In Fab1-HK129pAF, Fab9-HK129pAF and Fab10-HK129pAF, a second pAF incorporation position was added at the light chain position LL157 of the anti-CD3 Fab to generate new Fab molecules such as Fab1-HK129pAF-LL157pAF, Fab9-HK129pAF-LL157pAF and Fab10-HK129pAF, respectively. Fab10-HK129pAF-LL157pAF. This allows the use of any bifunctional linker as described in the previous examples to conjugate two folic acid plus two 5KPEG molecules in each Fab.

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

Figure 13A shows 5 µg of each purified CD3-folate bispecific antibody and 5K PEG conjugate. 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 folic acid, respectively. Lanes 5 and 8 represent conjugated 5KPEG-folate and bis-5KPEG-difolate, respectively.

Figure 13B shows the SDS-PAGE result 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 different bis-conjugated bis-5KPEG-difolate CD3 Fab compositions, and lanes 5 and 6 represent bis-conjugated bis10KPEG-difolate, respectively And 10KPEG-folic acid. The data (Figure 13A-B) shows the high purity (>90%) of all samples, where the expected molecular weight increases based on the PEG size.

In order to increase the conjugation efficiency, a two-step conjugation method was used to conjugate the purified CD3 Fab composition with PEG-folate. Use oxime chemistry to conjugate folate to the pAF position, then use maleimide-thiol click chemistry and 5K, 10K, or 20K PEG to conjugate PEG at the C-terminal cysteine of both the heavy and light chains . Figure 13C shows the SDS-PAGE of loading 10 μg protein per well under non-reducing (lane 2-5) and reducing (lane 7-10) conditions. Lanes 2 and 7 represent unconjugated difolate compositions, and lanes 3 and 8, 4 and 9, 5 and 10 respectively represent difolate-C-terminal bis-5KPEG, bis10KPEG and bis20KPEG compositions. The data in Figure 13C shows the high purity (>95%) of all samples, where the expected molecular weight increases based on the PEG size.

Example 19 :

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

表25 – 抗CD3 Fab1修飾的體外結合和細胞毒活性

Table 25-In vitro binding and cytotoxic activity of anti-CD3 Fab1 modification

In KB, OV-90 and SKOV-3 cells, in the presence of 50 nM SFA, CD3-folate bispecific antibody and 5K, 10K or 20K PEG C-terminal conjugates were also used for in vitro cytotoxicity studies (Figure 13D, Table 26). Although the potency decreased slightly with increasing PEG length, all the tested constructs retained effective cytotoxic activity. The results and trends are consistent across all cell lines tested. Based on these studies, it is speculated that the slight decrease in potency observed as the size of the PEG increases will be offset by the increased exposure due to the prolonged half-life.

表26 - 抗CD3-葉酸C末端修飾在各種細胞中的體外細胞毒性

Table 26-In vitro cytotoxicity of anti-CD3-folate C-terminal modification in various cells

表27比較了在存在20 nM SFA的情況下，親本Fab1以及兩個低親和力變體Fab 9和10與人類CD3的結合親和力。如所示，如以上實施例中所述，使用PEG-葉酸雙官能接頭完成的雙5KPEG-雙葉酸的共軛導致所有3個Fab與其未共軛對照相比的人類CD3結合降低和相應的細胞毒性潛力降低。

Table 27 compares the binding affinity of the parent Fab1 and the two low-affinity variants Fab 9 and 10 to human CD3 in the presence of 20 nM SFA. As shown, as described in the above example, the conjugation of bis-5KPEG-difolate using the PEG-folate bifunctional linker resulted in reduced human CD3 binding and corresponding cells for all 3 Fabs compared to their unconjugated control The toxicity potential is reduced.

表27 - 低親和力Fab 9和10的表徵

Table 27-Characterization of low affinity Fab 9 and 10

T cell activation and cytokine release: Table 27 shows that three modified Fabs activate the T cell marker CD69 and release two cytokines IFNγ and TNFα. As shown in the above example, even after significant modification by difolate-bis-5KPEG conjugation, the general correlation between CD3 binding strength, cytotoxic potential, T cell activation and subsequent cytokine release is in all Fabs Still established. This indicates 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. In addition, using proprietary UAA (unnatural amino acid) incorporation technology, position-specific PEGylation can improve PK properties, including half-life (T1/2) extension.

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 folic acid (physiologically relevant concentration of folic acid). The most effective CD3-folate bispecific antibody Fab1-HK129-LL157-difolate containing two folate molecules has 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 its potency was 31 times lower than that of Fab1-HK129-LL157-difolate. This data indicates that two folates can increase the efficacy of the CD3-folate bispecific antibody. The addition of 5KPEG reduces the 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. It was observed that the potency between Fab1-HK129-LL157-difolate and Fab1-HK129-LL157-bis-5KPEG-difolate with an IC50 value of 48.5 pM was reduced by 37.3 times. These data indicate that the CD3-folate bispecific antibody can maintain effective in vitro cytotoxicity at physiologically relevant concentrations of folic acid.

Example twenty one :

In vitro cytotoxicity data showed that in the presence of 20 or 50 nM folate, the CD3-folate bispecific antibody selectively killed SKOV3 cells expressing FOLRα. SKOV3 cells were treated with increasing concentrations of CD3-folate bispecific antibodies in 20 or 50 nM folic acid (physiologically relevant concentrations of folic acid) (Figures 15A and 15B). When the folic acid concentration was increased from 20 nM to 50 nM (close to the highest value of the normal physiologically relevant concentration of folic acid), the CD3-folate bispecific antibody showed a decrease in potency, ranging from 5.6 to 8 times. The addition of 5KPEG also resulted in a decrease in the efficacy of the CD3-folate bispecific antibody. A 5.4-fold decrease between monofolate and single 5KPEG folate bispecific antibody was observed, and a 22.9-fold decrease between difolate and double 5KPEG folate bispecific antibody. No significant difference in efficacy was observed between the monopegylated CD3-folate bispecific antibody and the dipegylated CD3-folate bispecific antibody. The data in Table 28 shows that in the presence of physiologically relevant concentrations of folic acid, the CD3-folate bispecific antibody can kill FOLRα-expressing cells, although the effectiveness of PEGylated antibodies is reduced compared with non-PEGylated antibodies. However, the CD3-folate bispecific antibody can kill cells expressing FOLRα.

表28 – SKOV3細胞中的體外細胞毒性

Table 28-In vitro cytotoxicity in SKOV3 cells

Example twenty two :

Further in vitro cytotoxicity studies were performed in the presence of 20 or 50 nM 5-mTHF. Treatment with increasing concentrations of CD3-folate bispecific antibody in the presence of 20 or 50 nM (physiologically relevant concentration of the main form of folic acid present in human serum) of 5-methyltetrahydrofolate (5-mTHF) SKOV3 cells (Figures 16A and 16B). The binding affinity of 5-mTHF to FOLRα is 1-10 nM, and the binding affinity to FOLRα is not as good as folic acid, which has a binding affinity of less than 1 nM. For the dual CD3-folate bispecific antibody and the single CD3-folate bispecific antibody, the CD3-folate bispecific antibody maintains a very effective IC50 value between 0.03 to 0.16 pM and 0.1 to 1.5 pM, respectively. The data in Table 29 indicate that in the presence of physiologically relevant concentrations of 5-mTHF, the CD3-folate bispecific antibody has effective in vitro cytotoxicity on SKOV3 cells expressing FOLRα.

表29 – 在5-mTHF存在下的體外細胞毒性

Table 29-In vitro cytotoxicity in the presence of 5-mTHF

Example twenty three :

Mouse pharmacokinetic studies were performed in CD1 mice: CD3-folate bispecific antibody was injected intravenously at a dose of 1 or 5 mg/kg via the mouse tail vein of CD-1 mice. Blood samples were collected at nine time points and analyzed via ELISA. The data clearly shows that the addition of 5KPEG increases serum exposure (AUClast) (Table 30-31 and Figure 17). Fab1-HK129-5KPEG-folate at 1 mg/kg showed a 4.3-fold increase over Fab1-HK129-folate, and Fab1-HK129-5KPEG-folate at 5 mg/kg showed a 5-fold increase over Fab1-HK129-folate. Fab1-HK129-LL157-bis-5KPEG-difolate showed a 16.25-fold increase compared to Fab1-HK129-LL157-difolate at 1 mg/kg, while Fab1-HK129-LL157-bis-5KPEG-difolate showed at 5 mg/kg It is 21.7 times higher than Fab1-HK129-LL157-Difolate. The data shows that there is a 3.9-fold difference between Fab1-HK129-5KPEG-folate and Fab1-HK129-LL157-bis-5KPEG-difolate. It was also observed that the serum half-life (T1/2) was improved 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. Compared with Fab1-HK129-LL157-difolate at 1 mg/kg, Fab1-HK129-LL157-bis-5KPEG-difolate showed a 4.2-fold increase in serum half-life, while Fab1-HK129-LL157-bis-5KPEG at 5 mg/kg -Difolate and Fab1-HK129-LL157-Difolate show a 6.25-fold increase in serum half-life. Data show that the addition of 5KPEG can improve the serum exposure and serum half-life of the CD3-folate bispecific antibody, which leads to a decrease in the frequency of administration to achieve effective serum exposure in the body.

表30 - CD3-葉酸雙特異性單琥珀抗體小鼠藥物代謝動力學分析

Table 30-CD3-folate bispecific mono-amber antibody pharmacokinetic analysis in mice

表31 - CD3-葉酸雙特異性雙琥珀抗體 (double Amber antibody)小鼠藥物代謝動力學分析

Table 31-CD3-folate double Amber antibody pharmacokinetic analysis in mice

Example twenty four

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

Study 1: To evaluate the effect of CD3-folate bispecific antibody on macrophages, the following experiments were performed: Fab1-HK129-LL157-pAF, Fab1-HK129-LL157-folic acid and Fab1-HK129-LL157-5KPEG folic acid were combined with Human M2 macrophages and human T cells are cultured together in the presence of 50 nM folic acid. Data show that Fab1-HK129-LL157-folate and Fab1-HK129-LL157-5KPEG-folate kill human M2 macrophages with IC50 values of 1.2 pM and 112.1 pM, respectively (data not shown). Fab1-HK129-LL157-pAF lacking folic acid does not cause the killing of human M2 macrophages. The data supports 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 produced from human blood from healthy donors and treated with granulocyte-macrophage colony stimulating factor (GM-CSF) for M1 macrophages Differentiation or treatment with macrophage colony stimulating factor (M-CSF) for M2 macrophage differentiation.

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

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

Effect of pegylation on CD3-folate bispecific antibodies: Table 32 shows the IC50 data of CD3-folate bispecific antibodies containing monofolate on macrophage cytotoxicity in vitro in the presence of 20 nM folic acid. The average IC50 of Fab1-HK129-folate in M1 macrophages is 85.0 pM (range 53.5-120.0 pM), and the average IC50 in M2 macrophages is 5.6 pM (range 1.4 pM-15.0 pM). According to the IC50 ratio, using Fab1-HK129-folate, M2 macrophages increased by an average of 26 times compared with M1 (range 5-42 times). Fab1-HK129-5KPEG-folate showed an average IC50 in M1 macrophages of 616.8 pM (range 198.0-908.9 pM), and an average IC50 in M2 macrophages of 13.4 pM (range 2.3-33.2 pM), where The improved average IC50 ratio between M1 and M2 macrophages was an average 69-fold increase in M2 macrophages (range 23-126-fold). These results indicate that the CD3-folate bispecific antibody containing monofolate has specificity for killing M2 macrophages, and the PEGylation of the CD3-folate bispecific antibody containing monofolate (Fab1-HK129-5KPEG-folate ) Compared with Fab1-HK129-folate, it is more selective for killing M2 macrophages.

表32 – 在20 nM葉酸存在下含單葉酸的CD3-葉酸雙特異性抗體對體外巨噬細胞的細胞毒性

Table 32-Cytotoxicity of CD3-folate bispecific antibodies containing monofolate to macrophages in vitro in the presence of 20 nM folic acid

The cytotoxicity results of in vitro macrophages of CD3-folate bispecific antibodies containing monofolate from a representative donor, Donor 6007, are depicted in Figure 18A. Arrows and numbers indicate the fold difference in IC50 between M1 and M2 macrophages. The dotted line represents Fab1-HK129-folate in M1 and M2, and the solid line represents Fab1-HK129-5KPEG-folate in M1 and M2.

Table 33 shows the IC50 data of the cytotoxicity of CD3-folate bispecific antibodies containing difolate on macrophages in vitro in the presence of 20 nM folic acid. The average IC50 of Fab1-HK129-LL157-difolate in M1 macrophages is 29.2 pM (range 10.9-42.8 pM) and the average IC50 in M2 macrophages is 1.5 pM (range 0.1 pM-5.5 pM), where The average IC50 ratio between M1 and M2 macrophages differed by 72 times (range 6-212 times). Fab1-HK129-LL157-Difolate-bis-5KPEG showed an average IC50 in M1 macrophages of 1620.8 pM (range 834.7-2651 pM) and an average IC50 in M2 macrophages of 15.7 pM (range 3.6-52.4 pM) ), where the average IC50 ratio between M1 and M2 macrophages was improved 181 times in M2 macrophages (range 49-501 times). These results indicate that the CD3-folate bispecific antibody containing difolate has specificity for killing M2 macrophages. Although the PEGylation of the CD3-folate bispecific antibody containing difolate (Fab1-HK129-LL157-Difolate-bis-5KPEG) was shown to be less effective, the selectivity for killing M2 macrophages was higher than that of Fab1- HK129-LL157-Difolate or Fab1-HK129-5KPEG-folate, as shown in Table XXX.

表33 – 在20 nM葉酸存在下含雙葉酸的CD3-葉酸雙特異性抗體對體外巨噬細胞的細胞毒性

Table 33-Cytotoxicity of CD3-folate bispecific antibodies containing difolate to macrophages in vitro in the presence of 20 nM folic acid

Figure 18B shows the cytotoxicity results of a CD3-folate bispecific antibody containing difolate from a representative donor, ie Donor 6007, on macrophages in vitro. Arrows and numbers indicate the fold difference in IC50 between M1 and M2 macrophages. The dotted line represents Fab1-HK129-LL157-Difolate in M1 and M2, and the solid line represents Fab1-HK129-LL157-Difolate-bis-5KPEG in M1 and M2.

Further studies were conducted to simulate the physiologically relevant concentrations of the main folate form present in human serum, which ranged between 9.1-45.1 nM. Within this physiological range, 86.7% (37.5 nM) is the main folate metabolite 5-methyl-tetrahydrofolate (5-mTHF), while only 4% (1.2 nM) is non-metabolized 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 is assumed that the concentration or composition of folic acid and 5-mTHF can affect the activity of the CD3-folate bispecific antibody. To evaluate this, the following experiment was performed: Table 34 and Table 35 show the cytotoxicity data of macrophages in vitro in the presence of 45 nM 5-mTHF. 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 average IC50 of Fab1-HK129-LL157-Difolate in M1 is 4.0 pM and the average IC50 in M2 is 0.05 pM, while the average IC50 of Fab1-HK129-LL157-Difolate-bis-5KPEG in M1 is 99.4 pM and The IC50 in M2 averaged 0.85 pM. 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 ratio between M1 and M2 of the CD3-folate bispecific antibody containing monofolate is an average of 43 times and 57 times, and the IC50 ratio between M1 and M2 of the CD3-folate bispecific antibody containing difolate is average It is 89 times and 212 times. These data indicate that bispecific antibodies containing CD3-folate can maintain the specific killing of M2 macrophages, and that PEGylation provides greater specificity at physiologically relevant 5-mTHF concentrations.

Table 34. Cytotoxicity of monofolate-containing CD3-folate bispecific antibodies to macrophages in vitro in the presence of 45 nM 5-mTHF

Table 35. Cytotoxicity of difolate-containing CD3-folate bispecific antibodies to macrophages in vitro in the presence of 45 nM 5-mTHF

Overall, the data indicate that in the presence of folic acid or 5mTHF metabolites, the di-pegylated CD3-folate composition shows greater resistance to M2 macrophages than the mono-pegylated CD3-folate composition. Specificity of killing. Therefore, an increase in selectivity is observed with more PEGylation.

Further studies on CD3-folate bispecific antibodies were also carried out in human myeloid derived suppressor cells (MDSC) to study FRβ expression. Treatment of human peripheral blood mononuclear cells (PBMC) from healthy donors with Fab1-HK129-5KPEG-folate and Fab1-LL157-bis-5KPEG-difolate to test its specificity against mononuclear MDSC (mMDSC) Cytotoxic activity of 0, 1, 10, and 100 pM. For each CD3-folate bispecific antibody, add it to PBMC and incubate in a 37°C and 5% CO2 incubator for 24 hours. After incubation, mMDSC was gated by the CD3-/CD33+/CD11b+/CD14+/HLA-DR low population using flow cytometry, and the percentage (%) of viable cells relative to the untreated control was measured.

Based on the observations, the percentage of mMDSC decreases 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-bis-5KPEG-difolate showed a dose-dependent reduction from 88% to 22% at 10 pM relative to 100 pM, respectively. These results indicate that treatment with CD3-folate bispecific antibody can selectively eliminate mMDSC as long as non-MDSC with high HLA-DR is retained.

In view of the fact that these suppressive cells can express folate receptors and participate in the inhibition of immuno-oncology drug activity, it is suggested that a combination therapy using checkpoint inhibitors and other immuno-oncology drugs can be used to target various cancers or diseases in which folate receptors can be expressed /Disease/Disorder treatment methods. This supports the use of the CD3-folate composition of the present 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 to FOLRa in several cell types. The binding affinity of CD3-folate bispecific antibody to KB cells expressing FOLRα: As shown in Table 36, Fab1-HK129-folate binds to KB cells expressing FOLRα with an affinity of 1.97 nM, and Fab1-HK129-5KPEG-folate has an affinity of 1.97 nM. 3.69 nM affinity binds to KB cells expressing FOLRa. Fab1-HK129-LL157-difolate binds to KB cells with an affinity of 0.85 nM, while Fab1-HK129-LL157-bis-5KPEG-difolate binds to KB cells with an affinity of 2.28 nM. Pegylated antibodies retain similar binding affinity to FOLRα-expressing KB cells as non-pegylated antibodies, which is in the low nM range.

表36 - CD3-葉酸雙特異性抗體與KB細胞的結合親和力

Table 36-Binding Affinities of CD3-Folic Acid Bispecific Antibodies to KB Cells

The binding affinity of the CD3-folate bispecific antibody to human T cells: Fab1-HK129-pAF (anti-CD3 Fab) does not contain folic acid or PEG, and binds to human T cells with an affinity of 1.59 nM (Table 37). Fab1-HK129-folate binds to human T cells expressing CD3 with an affinity of 1.53 nM, while Fab1-HK129-5KPEG-folate binds to human T cells expressing CD3 with an affinity of 2.34 nM. These CD3-folate bispecific antibodies have similar binding affinity to human T cells. Fab1-HK129-LL157-pAF binds to human T cells with an affinity of 0.604 nM. Fab1-HK129-LL157-difolate binds to human T cells with an affinity of 0.669 nM, while Fab1-HK129-LL157-bis-5KPEG-difolate has an affinity of 3.34 nM. These data indicate that the binding affinity of the pegylated CD3-folate bispecific antibody to human T cells is about 5 times lower than that of the non-pegylated antibody. These data also indicate that the CD3-folate bispecific antibody has a low nM binding affinity for human T cells.

表37 - CD3-葉酸雙特異性抗體與人類T細胞的結合親和力

Table 37-Binding Affinities of CD3-Folic Acid Bispecific Antibodies to Human T Cells

Binding affinity of CD3-folate bispecific antibody to cynomolgus T cells: As shown in Table 38, Fab1-HK129-pAF binds to cynomolgus T cells with an affinity of 2.67 nM. Fab1-HK129-folate binds to cynomolgus T cells expressing CD3 with an affinity of 2.69 nM, while Fab1-HK129-5KPEG-folate binds with an affinity of 3.31 nM. This indicates that the CD3-folate bispecific antibody with a single amber position has similar binding affinity to cynomolgus monkey T cells. The CD3-folate construct with double amber position showed similar binding affinity in cynomolgus monkey T cells. As shown in Table 38, Fab1-HK129-LL157-pAF binds to cynomolgus T cells with an affinity of 0.995 nM. Fab1-HK129-LL157-Difolate binds to cynomolgus T cells with an affinity of 1.0 nM, and Fab1-HK129-LL157-Double 5KPEG-Difolate binds to cynomolgus T cells with an affinity of 5.01 nM. Therefore, the data further indicate that the binding affinity of the pegylated CD3-folate bispecific antibody to cynomolgus monkey T cells is about 5 times lower than that of the non-pegylated CD3-folate bispecific antibody. Overall, the data indicate that the CD3-folate bispecific antibody has a low nM binding affinity to cynomolgus monkey T cells.

表38 - CD3-葉酸雙特異性抗體與食蟹猴T細胞的結合親和力

Table 38-Binding affinity of CD3-folate bispecific antibodies to cynomolgus monkey T cells

Example 26 :

The following study illustrates the in vivo safety and efficacy study of the CD3 folate antibody of the present invention in mice.

Study 1: In female immunocompromised mice bearing human cervical tumors derived from KB cell lines, multiple doses were administered to test the anti-tumor efficacy of the anti-CD3-folate bispecific antibody. A non-targeted anti-CD3 antibody was included as a control. Female NSG mice with KB cervical tumors (2.0×106 cells inoculated from the 7th generation, and initially grown from the frozen 3rd generation) were divided into 5 groups, each with 8 animals. When the size of the tumor was about 100 mm3, 7.5×106 PanT cells were inoculated into mice intraperitoneally. After 24 hours, the mice were randomly divided into the following treatment groups: G1: anti-CD3 Fab1-HK129-pAF control (0.05 mpk), G2 and G3 each had 0.01 mpk and 0.05 mpk CD3 Fab1-HK129-LL157-difolate -Double 5KPEG, G4: Fab9-HK129-LL157-Difolate-Double 5KPEG (0.125 mpk), and G5: CD3 Fab1-HK129-folate control (0.25 mpk); and determine tumor volume (Figure 19A) and quality/weight ( Figure 19B). When the tumor was approximately ~125 mm3 on average, all mice were administered intravenously (IV) on day 7. The animals were monitored for tumor growth (measured by calipers) and body weight twice a week. As shown in Figure 19A, all CD3-folate bispecific compositions showed efficacy, among which group 3 (Fab1-HK129-LL157-difolate-bis-5KPEG, 0.05 mpk) had the greatest effect on inhibiting tumor growth (TGI = 82%). Except for the control CD3 Fab-HK129-pAF antibody, all compositions caused weight loss (Figure 19B).

Biomarker analysis: blood samples were taken from each animal before the start of treatment and on the 7th and 24th days after the start of treatment. The percentage of human CD45/mouse CD45 in the sample was analyzed by FAC analysis to determine whether the treatment increased the peripheral human T cell population as proposed by CD3-folate activation/targeting. At the end of the study, the human lymphocyte marker hCD45 was used to analyze the presence of tumor infiltrating lymphocytes (TIL) in the tumor by FAC. All CD3-folate treatment groups have shown anti-tumor efficacy and have the ability to increase the blood level of human CD45 to varying degrees. Among them, human CD45 has the highest level and is attributed to the 0.05 mpk bispecific antibody Fab1-HK129-LL157-double Folic acid-bis-5KPEG treatment (Figure 19C). This study also examined tumor growth inhibition and the ability of the CD3-folate composition of the present invention to promote tumor infiltrating lymphocytes (TIL). In addition, it was found that the presence of TIL (a marker of tumor immune surveillance activation) was increased in all treatment groups compared to the control group, and the third group (Fab1-HK129-LL157-difolate-bis-5KPEG, 0.05 mpk) had The highest TIL induction (Figure 19D).

Study 2: This study examined changes in tumor growth inhibition (TGI) and human CD45 percentages in mouse blood/tumor infiltration in NCG mice bearing KB tumors. Single (Fab1-HK129-LL157-folate-5KPEG) and double (Fab1-HK129-LL157-difolate- The anti-tumor efficacy of double 5KPEG) PEGylated anti-CD3-folate bispecific antibody. A non-targeting CD3 antibody was included as a control. Thirty-five female NCG mice with KB cervical tumors (inoculated with 2.0×106 cells from the 5th generation, and initially grew from the frozen 3rd generation) were divided into 3 groups, each with 10 animals. When the tumor is about 85-100 mm3, mice are 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-difolate-bis-5KPEG, Each group was 0.025 mpk every 5 days (Figure 20A and 20B). On the 7th day, all mice were inoculated with 6.0×106 Pan-T cells and administered intravenously (IV) on the 8th day. At this time, the average tumor size was about 100 mm3. Animals were monitored for tumor growth (Figure 20A) (measured by calipers) and body weight (Figure 20B) twice a week. All CD3-folate targeting compositions show different degrees of efficacy, among which G2: Fab1-HK129-LL157-folate-5KPEG (single PEG composition) has the greatest impact on the growth of tumors (TGI = 85%), and 5 out of 10 mice caused complete tumor regression (CR). In contrast, G3: Fab1-HK129-LL157-difolate-bis-5KPEG (double PEG composition), its TGI was 76%, and 10 mice Three of the animals had CR (Figure 20A). All tested substances were tolerated, and G3 (double PEG composition) showed slight weight loss in several mice, but these weight loss did not exceed 15% (Figure 20B).

Biomarker analysis: Blood samples were taken from each animal on the 7th and 14th days after the start of the above-mentioned treatment. Analyze the percentage change of human CD45 in blood samples to determine whether the treatment increases the peripheral human T cell population through CD3-folate activation/targeting. At the end of the study, the human lymphocyte marker hCD45 was used to analyze tumor infiltrating lymphocytes (TIL) in the tumor by FAC. All CD3-folate treatment groups showed anti-tumor efficacy and had the ability to increase human CD34 levels in blood (Figure 20C) and tumors (Figure 20D) to varying degrees. Among them, the highest levels of human CD45 and TGI were attributed to the use of single drugs. Pegylated CD3-folate targeting composition therapy. Study 3: Shows similar results to Study 1 and Study 2. Fifty NSG humanized female mice (CD34+ from Jackson laboratories) with KB cervical tumors (2.0×106 cells inoculated from the 5th generation, and initially grown from the frozen 3rd generation) were divided into 5 Group, 10 animals in each group. When the tumor is about 80-100 mm3, treatment is started. G1: Fab1-HK129-pAF control (0.0125 mpk), G2: Fab1-HK129-difolate (0.0025 mpk), G3: Fab1-HK129-difolate (0.0125 mpk), G4: Fab1-HK129-difolate-bis-5KPEG (0.0025 mpk), G5: Fab1-HK129-Difolate-bis-5KPEG (0.0125 mpk). All mice were administered intravenously (IV) twice a week. Animals were monitored for tumor growth (Figure 21A) (measured by calipers) and body weight (Figure 21B) twice a week. On the 5th day of treatment (the first day after the second dose), blood samples drawn from the animals were analyzed for T cell activation. The percentages of human CD45, CD3, CD25 and CD69 in blood samples were analyzed by FAC analysis to determine whether the CD3-folate targeting composition increased the expression of T cell activation markers (Figure 21C-21F). At the end of the study, tumor infiltrating lymphocytes (TIL) were also analyzed, in which TIL (CD45, CD3, and CD8) analysis was performed on 5 tumors in each group by FAC. All CD3-folate targeting compositions showed varying degrees of efficacy. However, at similar doses, all PEGylated CD3-folate targeting compositions (TGI of the G4 group was 87%, and the TGI of the G5 group was 91 %) are superior to its non-pegylated isoforms at the same dose (TGI of G2 group is 51%, TGI of G3 group is 67%), including induction of T cell activation marker CD25/CD69, increasing TIL, and has a greater anti-tumor growth inhibitory effect. All test substances caused a slight weight loss in mice, but did not exceed 15%.

Study 4: Testing the CD3-folate bispecific antibodies Fab1-HK129-LL157-Difolate and Fab1-HK129-LL157 by multi-dose administration in female immunocompromised mice bearing human cervical tumors derived from the KB cell line -Difolate-the anti-tumor effect of double 5KPEG. A non-targeting CD3 antibody (Fab1-HK129-pAF) was included as a control. Sixty-five female NSG mice with KB cervical tumors (inoculated with 2.0×106 cells from the 7th generation and initially grown from the frozen 3rd generation) were divided into 6 groups, each with 10 animals. When the tumor is about 85-100 mm3, 7.5×106 PanT cells are intraperitoneally inoculated 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-difolate-double 5KPEG (0.0125 mpk), G3: Fab1-HK129-LL157- Difolate-bis-5KPEG (0.05 mpk), G4: Fab1-HK129-LL157-difolate (0.0125 mpk), G5: Fab1-HK129-LL157-difolate (0.05 mpk). When the tumors averaged approximately ~100 mm3, all mice were given intravenous (IV) administration on the 8th day. The animals were monitored for tumor growth (measured by calipers) and body weight twice a week. As shown in Figure 22A, all CD3-folate targeting compositions showed anti-tumor activity (TGI=G2:92%, G4:55%, G5:90%), of which the G3 group (Fab1-HK129-LL157-Difolate -Double 5KPEG, 0.05 mpk) had the greatest effect on hindering tumor growth (TGI = 99%), and caused 6 out of 8 mice to experience complete tumor regressions (CR). With the exception of the control CD3 Fab antibody, all test compositions caused some weight loss, but no more than 15% loss (Figure 22B). The results of this study indicate a dose dependent response, which can be attributed to the biophysical properties of adding PEG to the CD3 Fab-folate compound.

Study 5: Testing the CD3-folate bispecific antibodies Fab1-HK129-LL157-Difolate and Fab1-HK129 by multiple dose administration in female immunocompromised mice bearing human ovarian tumors derived from the OV-90 cell line -The anti-tumor efficacy of LL157-Difolate-bis-5KPEG (Figure 23A and 23B). OV-90 was selected to determine whether the CD3 Fab-folate composition of the present invention is active in a model that is resistant to the standard of care for ovarian cancer, namely platinum-based therapy. A non-targeting CD3 antibody (Fab1-HK129-pAF) was included as a control. Seventy female NSG mice with OV-90 ovarian tumors (inoculated with 7.5×106 cells from the 5th passage, initially growing from the frozen 2nd passage) were divided into 6 groups, each with 10 animals. When the tumor is about 100-200 mm3, 7.5×106 PanT cells were inoculated into mice intraperitoneally. After 24 hours, the mice were randomly divided into the following treatment groups: G1: Fab1-HK129-pAF control, G2: Fab1-HK129-LL157-difolate (0.0125 mpk), G3: Fab1-HK129-LL157-difolate (0.05 mpk), G4: Fab1-HK129-LL157-difolate-bis-5KPEG (0.0125 mpk), G5: Fab1-HK129-LL157-difolate-bis-5KPEG (0.05 mpk), and G6: carboplatin (100 mpk). When the tumors averaged approximately ~100 mm3, all mice were given intravenous (IV) administration on day 8. 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. The animals were monitored for tumor growth (measured by calipers) and body weight twice a week. Although the OV-90 model resists the standard of care for carboplatin treatment of ovarian cancer (group G6), all CD3-Fab folate targeting compositions tested showed varying degrees of efficacy, among which group G5 (Fab1-HK129-LL157 -Difolate-bis-5KPEG, 0.05 mpk) had the greatest effect on hindering tumor growth (TGI = 81%); Figure 23A. Except for the CD3 Fab antibody in the control group, 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-difolate-bis-5KPEG had better results than the group treated with Fab1-HK129-LL157-difolate Good anti-tumor activity. This can be attributed to the enhancement of biophysical properties caused by the addition of PEG to the CD3 Fab-folate composition. It should also be noted that in ovarian cancer cells with a low copy number of folate receptors (for example, OV-90 with ~10K copies per cell), strong anti-tumor effects are still observed. This supports the use of the CD3 Fab-folate composition of the present invention as a first-line therapeutic agent for the treatment of patient populations without the prerequisite of high folate receptor copy number. The in vitro cytotoxicity analysis of the present invention further confirmed this point, indicating that cells with low folate receptor copy numbers still have EC50 values in the picomolar range.

Example 27 :

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

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

Patients: 18 healthy volunteers were included in the study, ranging in age from 20-40 years old and weighing between 60-90 kg. The subject will not have clinically significant abnormal laboratory values in hematology or serum chemistry, and will be negative for urine toxicology screening, HIV screening, and hepatitis B surface antigen. They should not have any of the following signs: high blood pressure; history of any primary blood disease; history of major liver, kidney, cardiovascular, gastrointestinal, genitourinary, metabolic, and neurological diseases; history of anemia or seizures; Known sensitivity of mammalian-derived products, PEG or human serum albumin; habitual and heavy consumers of caffeine-containing beverages; participating in any other clinical trials or conducting blood transfusions or donations within 30 days of entering the study; Has been exposed to anti-CD3 antibodies within three months of the study; became ill within 7 days of entering the study; and there was a significant abnormality in the pre-study physical examination or clinical laboratory evaluation within 14 days of entering the study. The safety of all subjects can be assessed and all blood collections for pharmacokinetic analysis are collected as planned. All studies were conducted with the approval of the institutional ethics committee and the patient's consent.

This will be a Phase I, single-center, open label, and randomized two-cycle crossover study in healthy female volunteers. Eighteen subjects were randomly assigned to one of two treatment sequence groups (nine subjects/group). The anti-CD3 antibody is administered by a person skilled in the art through any route usually used to introduce the antibody into a subject in need, via a local or systemic administration period. The anti-CD3 antibody composition according to the present invention can be administered subcutaneously, intramuscularly, intracutaneously, intraarticularly, intrapleurally, intraperitoneally, intraarterially, or intravenously, but the most suitable route in any given situation will depend on the application. The nature and severity of the disease and/or condition being treated. For example, it can be administered in large doses subcutaneously (sc) or intravenously in two separate administration periods, using equivalent doses of bispecific anti-CD3 Fab-folate antibodies containing non-naturally encoded amino acids and all Selected commercially available products. The dosage and frequency of administration of the commercially available products are in accordance with the instructions on the package label. Additional dosing, dosing frequency, other required parameters, and use of commercially available products can be added to the study by including other groups of subjects. Each dosing period is separated by a washout period of 14 days. For each of the two dosing periods, the subject is restricted to the research center at least 12 hours before the dosing and 72 hours after the dosing, but there is no restriction between the dosing periods. If additional dosing, frequency, or other parameters need to be tested on the pegylated bispecific anti-CD3 antibody, other groups of subjects can be added. The experimental preparation of the anti-CD3 antibody is a bispecific anti-CD3 Fab-folate antibody containing a non-naturally encoded amino acid.

Blood sampling: Before and after administration of the anti-CD3 Fab-folate bispecific antibody, blood was continuously drawn by direct venipuncture. About 30, 20, and 10 minutes before dosing (3 baseline samples) and about the following time 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 concentration. Each serum sample is divided into two aliquots. All serum samples are stored at -20°C. Serum samples are shipped on dry ice. Fasting clinical laboratory tests (hematology, serum chemistry, and urinalysis) were performed immediately before the initial dosing on day 1, in the morning on day 4, before dosing on day 16, and in the morning on day 19.

Biological analysis method: Use radioimmunoassay (RA) or ELISA kit program to determine the serum bispecific anti-CD3 Fab-folate antibody concentration.

Safety determination: Record vital signs before each administration (day 1 and 16) and 6, 24, 48 and 72 hours after each administration. Safety determination is based on the incidence and type of adverse events and the change from baseline in clinical laboratory tests. In addition, changes in vital sign measurements compared to before the study were evaluated, including blood pressure and physical examination results.

Data analysis: By subtracting the average baseline anti-CD3 antibody concentration from each post-administration value, the serum concentration value after administration is corrected relative to the baseline anti-CD3 antibody concentration before administration, the average baseline anti-CD3 antibody concentration It was determined by averaging the levels of anti-CD3 antibodies from three samples collected 30, 20, and 10 minutes before administration. If the serum anti-CD3 antibody concentration before administration is lower than the determined quantitative level, it is not included in the average calculation. The pharmacokinetic parameters were determined based on serum concentration data corrected for the baseline anti-CD3 antibody concentration. The latest version of the BIOAVL software was used to calculate the pharmacokinetic parameters through a model-independent method on the Digital Equipment Corporation VAX 8600 computer system. Determine the following pharmacokinetic parameters: peak serum concentration (Cmax); time to reach peak serum concentration (tmax); area under the concentration-time curve (AUC) from time zero to the last blood collection time calculated using the linear trapezoidal rule ( AUC0-72); and the terminal elimination half-life (t1/2) calculated based on the elimination rate constant. The elimination rate constant is estimated by linear regression of continuous data points in the linear region at the end of the log-linear concentration-time graph. Calculate the average value, standard deviation (SD) and coefficient of variation (CV) of the pharmacokinetic parameters for each treatment. Calculate the ratio of the parameter average (preserved preparation/non-preserved preparation).

Safety results: The incidence of adverse events was evenly distributed among the treatment groups. Compared with baseline or pre-study clinical laboratory tests or blood pressure, there were no clinically significant changes, and there were no significant changes in physical examination results and vital sign measurements compared to pre-study. The safety profiles of the two treatment groups should look similar.

Pharmacokinetic results: The average serum anti-CD3 antibody Fab-folate bispecificity of all 18 subjects after receiving a single dose of one or more commercial products specific to the same target antigen at each measurement time point Concentration-time curves (not yet corrected for baseline anti-CD3 antibody levels) were compared with bispecific anti-CD3 antibodies containing non-naturally encoded amino acids. The baseline anti-CD3 antibody concentration before administration of all subjects should be within the normal physiological range. The pharmacokinetic parameters were determined based on the serum data corrected for the average baseline anti-CD3 antibody concentration before administration, and the Cmax and tmax were determined. The average tmax of the selected clinical comparators is significantly shorter than the tmax of the bispecific anti-CD3 antibody containing a non-naturally encoded amino acid. Compared with the terminal half-life of the bispecific anti-CD3 antibody containing a non-naturally encoded amino acid of the present invention, the terminal half-life value of the tested commercially available anti-CD3 antibody product is significantly shorter.

In conclusion, a single dose of a bispecific anti-CD3 antibody containing a non-naturally encoded amino acid administered subcutaneously 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 patients and healthcare providers with tremendous clinical utility.

It should be understood that the embodiments and embodiments described in the present invention are only for illustrative purposes, and various modifications or changes in view of them will be conceived by those skilled in the art and will be included in the spirit and scope of the present application and the attached patent application. Within the scope of the range. All publications, patents, and patent applications cited in the present invention are incorporated by reference in their entirety into the present invention for all purposes.

The invention is further described by the following numbered examples:

1. An anti-CD3 Fab antibody comprising at least one of SEQ ID NO: 1-61.

2. The anti-CD3 Fab antibody described in the scope of the patent application 1, which comprises two of SEQ ID NOs: 1-61.

3. A bispecific binding molecule comprising i) a first binding domain and ii) a second binding domain, wherein the second binding domain is selected from SEQ ID NO: 1-61.

4. A cytotoxic active CD3 Fab specific binding construct, which comprises the amino acid sequence shown in one or more of SEQ ID NO: 1-61.

5. An anti-CD3 Fab antibody, wherein the anti-CD3 antibody comprises a binding domain, the binding domain comprising (a) a VH domain, the VH domain comprising an amine group selected from SEQ ID NO: 1-3 Acid sequence, and (b) a VL domain comprising an amino acid sequence selected from SEQ ID NO: 4-5.

6. The anti-CD3 Fab antibody as described in embodiment 4, wherein the antibody is linked to a linker, polymer or biologically active molecule.

7. The anti-CD3 antibody of embodiment 6, wherein the biologically active molecule is folic acid.

8. The anti-CD3 Fab antibody as described in Example 1, which incorporates a non-naturally encoded amino acid.

9. The anti-CD3 Fab variant as described in embodiment 6, wherein the heavy chain further comprises an amino acid extension at the C-terminus.

10. The anti-CD3 Fab variant of embodiment 9, wherein the amino acid extension comprises the amino acid DKTHT.

11. The anti-CD3 Fab antibody of embodiment 5, wherein the heavy chain and light chain sequences contain framework residues or germline mutations at one or more antigen binding positions.

12. The anti-CD3 Fab antibody as described in embodiment 6, wherein the linker is a water-soluble polymer.

13. The anti-CD3 Fab antibody of embodiment 12, wherein the water-soluble polymer is linear or branched.

14. The anti-CD3 Fab antibody of embodiment 6, which comprises one or more folic acid and one or more polyethylene glycol.

15. A method for optimizing cell killing in cells expressing a high number of folate receptors, the method comprising the anti-CD3 Fab antibody as described in Patent Scope 5, wherein the antibody comprises one or more folic acid; and One or more non-naturally encoded amino acids are incorporated into the antibody.

16. The method of embodiment 15, wherein the number of folate receptors is equal to or greater than 10,000.

17. The anti-CD3 Fab antibody as described in embodiment 5, which further comprises a PEG folate linker having a structure according to compound 30.

18. A method of treating a patient with a disease or disorder in cells expressing a high folate receptor, the method comprising administering to the patient a therapeutically effective amount of an anti-CD3 Fab antibody as described in the scope of the patent application 17.

19. The bispecific anti-CD3 Fab antibody as described in Example 3, which contains two folic acid and two PEGylated molecules.

20. The bispecific anti-CD3 Fab antibody as described in Example 19, which further comprises a non-naturally encoded amino acid that is position-specifically incorporated.

CD25 +: T cell activation marker positive CD69 +: T cell activation marker positive C _H 1: heavy chain constant region C _L: light chain constant region Fab: antigen binding region Fab 1 ~ 14 Fab: antigen binding region Segment 1~14 Fab 16: Antigen binding segment 16 Fab 17: Antigen binding segment 17 Fab 21: Antigen binding segment 21 Fab1-HK129: Heavy chain variant of engineered anti-CD3 Fab Fab1-HK129-pAF: The heavy chain pAF variant of the engineered anti-CD3 Fab Fab1-HK129-LL157-pAF: the heavy chain-light chain (dual) pAF variant of the engineered anti-CD3 Fab CD3 (HK129pAF): the engineered anti-CD3 Fab CD3 heavy chain pAF variant CD3 (HK129pAF-LL157pAF): engineered anti-CD3 Fab heavy chain-light chain (double) pAF variant 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: polyethylene glycol of molecular x SDS-PAGE: polyacrylamide gel electrophoresis T1 / 2: half life TIL: tumor infiltrating lymphocytes V _H: heavy chain variable region V _L: light chain variable region

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

Figures 2A-2F depict the titration of humanized anti-CD3 Fab bound to human and cyno PBMC. (Figure 2A), Fab1 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 the rat pharmacokinetic (PK) analysis of the anti-CD3 Fab1 molecule. Pegylation of the humanized anti-CD3 Fab1 molecule can prolong the plasma half-life (T1/2) in rats.

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

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

Figures 6A-6B depict the 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 low-affinity variants of anti-CD3 Fab-HK129-folate molecules purified from E. coli cells binding 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 cyno CD3. Figure 7A shows the first round of binding, and Figure 7B shows the second round of low affinity variants of anti-CD3 Fab-HK129-folate molecules purified from E. coli cells binding to cynomolgus CD3. Fab1-HK129-folate (parental control) was included as a positive control.

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

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

Figures 10A-10B depict the activation of T cells. Figure 10A shows the activation of 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 effects of various low-affinity anti-CD3 Fab-HK129-folate variants in the absence of SKOV-3 tumor cells (Figures 11A and 11C) or 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 the anti-CD3 Fab-folate bispecific conjugate and PEG linker of the present invention. Figure 12A is a representative example of folate-PEG ligand (ligand); Figure 12B is a representative example of folate-branched PEG ligand; Figure 12C-12D is a representative of CD3-folate bispecific antibody and PEG conjugates Figure 12E is a representative diagram of a CD3-folate bispecific antibody with C-terminal pegylation; and Figure 12F is a cross-linking of CD3-folate bispecific antibodies with C-terminal PEGylation by CD3-Fab Representative description 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 the anti-CD3 Fab-folate bispecific antibody selectively kills FOLRα-expressing KB cells.

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

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

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 anti-CD3 Fab-folate bispecific antibodies containing monofolate to macrophages in vitro. Arrows and numbers indicate the fold difference in IC50 between M1 and M2 macrophages. The dotted line represents Fab1-HK129-folate in M1 and M2, and the solid line represents Fab1-HK129-5KPEG-folate in M1 and M2. Figure 18B shows the cytotoxicity of anti-CD3 Fab-folate bispecific antibodies containing difolate to macrophages in vitro. Arrows and numbers indicate the fold difference in IC50 between M1 and M2 macrophages. The dotted line represents Fab1-HK129-LL157-Difolate in M1 and M2, and the solid line represents Fab1-HK129-LL157-Difolate-bis-5KPEG 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 quality/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 quality/body weight; Figures 20C and 20D show human CD45 expression and TIL induction, respectively.

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

N: Nitrogen atom

O: oxygen atom

PEGx: x molecules of polyethylene glycol

Claims (44)

An anti-CD3 antibody or antibody fragment comprising the amino acid sequence of at least one of SEQ ID NO: 1-62. The anti-CD3 antibody or antibody fragment according to claim 1, which comprises the amino acid sequence of both of the SEQ ID NOs: 1-62. The anti-CD3 antibody or antibody fragment according to claim 1, which comprises: (a) The 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 (b) The SEQ ID NO: any one of 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. The anti-CD3 antibody or antibody fragment according to claim 3, which comprises any one of the SEQ ID NO: 1-6 and any one of the SEQ ID NO: 7-9. The anti-CD3 antibody or antibody fragment according to claim 4, which comprises any one of the SEQ ID NO: 4-6 and any one of the SEQ ID NO: 7-9. The anti-CD3 antibody or antibody fragment according to any one of claims 1 to 5, wherein the antibody or antibody fragment comprises IgG, Fv, Fab, (Fab')2 or single chain Fv (scFv). A bispecific binding molecule comprising: (1) A first binding domain; and (2) A second binding domain, wherein the second binding domain comprises at least one of the SEQ ID NOs: 1-62. The bispecific binding molecule according to claim 7, wherein the second binding domain comprises two of the SEQ ID NO: 1-62. The bispecific binding molecule according to claim 7, wherein the second binding domain comprises: (a) The 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 (b) The SEQ ID NO: 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. The bispecific binding molecule according to claim 7, wherein the second binding domain comprises any one of the SEQ ID NO: 1-6 and any one of the SEQ ID NO: 7-9. A cytotoxic activity CD3 specific binding construct comprising at least one of the SEQ ID NO: 1-62. A cytotoxic activity CD3 specific binding construct comprising two of the SEQ ID NO: 1-62. A cytotoxic activity CD3 specific binding construct, which comprises: (a) 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 Any of, 54, 55, 56 and 57; and (b) SEQ ID NO: any one of 7, 8, 9, 18, 19, 20, 39, 58, 59, 60, 61, and 62. The cytotoxic active CD3 specific binding construct according to claim 13, which comprises any one of the SEQ ID NO: 1-6 and any one of the SEQ ID NO: 7-9. The anti-CD3 antibody or antibody fragment according to any one of claims 1-6, wherein the antibody or antibody fragment comprises one or more non-naturally encoded amino acids, wherein the non-naturally encoded amino acid is para-acetyl Para-acetyl phenylalanine, p- nitrophenylalanine, p- sulfotyrosine, p- carboxyphenylalanine, o-nitrophenylalanine o- nitrophenylalanine), m- nitrophenylalanine, p- boronyl phenylalanine, o - boronyl phenylalanine, m - boronyl phenylalanine ), of amino acid amphetamine (p -aminophenylalanine), amphetamine anthranilic acid (o -aminophenylalanine), between amino phenylalanine (m -aminophenylalanine), of acyl-phenylalanine (p -acylphenylalanine), o-acyl methamphetamine acid (o -acylphenylalanine), between the acyl phenylalanine (m -acylphenylalanine), on OMe phenylalanine (p -OMe phenylalanine), o-OMe phenylalanine (o -OMe phenylalanine), inter-OMe phenylalanine (m -OMe phenylalanine) , P- sulfophenylalanine (p-sulfophenylalanine), o-sulfophenylalanine ( o- sulfophenylalanine), m-sulfophenylalanine ( m- sulfophenylalanine), 5-nitrohistidine (5-nitro His), 3- 3-nitro Tyr, 2-nitro Tyr, nitro substituted leucine (nitro substituted Leu), nitro substituted histidine (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-nitr o Trp), 6-nitro Trp (6-nitro Trp), 7-nitro Trp (7-nitro Trp), 3-aminotyrosine, 2-aminotyrosine (2 -aminotyrosine), O- sulfotyrosine (o -sulfotyrosine), 2-sulfooxyphenylalanine (2-sulfooxyphenylalanine), 3-sulfooxyphenylalanine (3-sulfooxyphenylalanine), o-carboxyphenylalanine ( o- carboxyphenylalanine), between the carboxyl phenylalanine (m -carboxyphenylalanine), acetylsalicylic -L- phenylalanine (p -acetyl-L-phenylalanine) , on to propargyl - phenylalanine (p -propargyl-phenylalanine), O- methyl -L-tyrosine ( o- methyl-L-tyrosine), L-3-(2-naphthyl)alanine (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 (4-propyl-L-tyrosine), tri-O- acetyl-GlcNAcβ-serine (tri-o- acetyl-GlcNAcβ-serine), L-dopa (L-DOPA), fluorine Fluorinated phenylalanine, isopropyl-L-phenylalanine, p- azido-L-phenylalanine, p-azido-L-phenylalanine, p-azido-L-phenylalanine Acid ( p- acyl-L-phenylalanine), p- benzoyl-L-phenylalanine (p-benzoyl-L-phenylalanine), L-phosphoserine (L-phosphoserine), phosphonoserine ), phosphonotyrosine, p- iodo-phenylalanine, p- bromophenylalanine, p-amino-L-phenylalanine ), isopropyl-L-phenylalanine (isopropyl-L-ph enylalanine) and p- propargyloxy-L-phenylalanine. The anti-CD3 antibody or antibody fragment according to claim 15, wherein the non-naturally encoded amino acid is specifically incorporated into the antibody by position. The anti-CD3 antibody or antibody fragment according to any one of claims 1-6 and 15, wherein the anti-CD3 antibody or antibody fragment is linked to a linker, a polymer or a biologically active molecule. The anti-CD3 antibody or antibody fragment according to claim 17, wherein the linker or the polymer is a bifunctional polymer or a bifunctional linker. The anti-CD3 antibody or antibody fragment according to any one of claims 17 and 18, wherein the biologically active molecule comprises a small molecule. The anti-CD3 antibody or antibody fragment according to any one of claims 1-6, 15 and 17-19, wherein the biologically active molecule comprises one or more folate molecules or analogs or derivatives thereof. The anti-CD3 antibody or antibody fragment according to any one of claims 1-6, 15 and 17-20, wherein the non-naturally encoded amino acid is linked to a water-soluble polymer. The anti-CD3 antibody or antibody fragment according to claim 21, wherein the water-soluble polymer comprises one or more polyethylene glycol (PEG). The anti-CD3 antibody or antibody fragment of claim 22, wherein the PEG is linked to the non-naturally encoded amino acid or the biologically active molecule. The anti-CD3 antibody or antibody fragment according to claim 22, 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 24, 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 of any one of claims 22-25, wherein the PEG is linear or branched. The anti-CD3 antibody or antibody fragment according to claim 17, which comprises one or more folic acid and one or more PEG molecules. The anti-CD3 or antibody fragment according to claim 3, wherein the heavy chain and the light chain sequence comprise framework residues and/or germline mutations at one or more antigen binding positions. The anti-CD3 antibody or antibody fragment according to claim 28, which is used for antigen binding optimization. The anti-CD3 antibody or antibody fragment according to claim 17, which further comprises 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 according to claim 7, wherein the antibody or the antibody fragment comprises one or more post-translational modifications. The anti-CD3 antibody or antibody fragment as described in any one of claims 1-6 and 15-31, the bispecific binding molecule as described in any one of claims 7-10, or as claimed in claims 11-14 The cytotoxic activity CD3 specific binding construct of any one of which is used in therapy. A method for optimizing cell killing in cells expressing high folate receptor numbers, wherein the method comprises the anti-CD3 antibody or antibody fragment as described in any one of claims 1-6 and 15-31, and as claimed The bispecific binding molecule according to items 7-10, or the cytotoxic active CD3 specific binding construct according to claims 11-14, wherein the anti-CD3 antibody, the bispecific binding molecule or the cytotoxic activity The CD3 specific binding construct contains one or more folic acid, and one or more non-naturally encoded amino acids are incorporated into the antibody. The method according to claim 33, wherein the number of folate receptors is equal to or greater than 10,000. The method according to claim 33, which further comprises C with one or more non-naturally encoded amino acids or one or more folic acid molecules or the C-terminus of the heavy chain or the light chain or both the heavy chain and the light chain. One or more PEG molecules attached to the ends. A method of treating a subject having a disease or disorder in a cell expressing a high folate receptor, wherein the method comprises administering to the patient a therapeutically effective amount of any one of claims 1-6 and 15-31 Anti-CD3 antibody. A method of treating a patient suffering from cancer, wherein the method comprises administering to the patient a therapeutically effective amount of the anti-CD3 antibody according to any one of claims 1-6 and 15-31. The method of claim 37, wherein the cancer is characterized by high expression of folate receptor alpha (FOLR1), and wherein, optionally, the cancer is an ovarian cancer. The method according to claim 38, wherein the ovarian cancer comprises an epithelial tumor, a mesenchymal tumor, a germ cell tumor, a fallopian tube cancer, or a primary peritoneal cancer. A method of treating a patient suffering from diabetes, wherein the method comprises administering to the patient a therapeutically effective amount of the anti-CD3 antibody according to any one of claims 1-6 and 15-31. A method of treating a patient suffering from AIDS, wherein the method comprises administering to the patient a therapeutically effective amount of the anti-CD3 antibody according to any one of claims 1-6 and 15-31. A method for treating a patient suffering from a genetic disease, wherein the method comprises administering to the patient a therapeutically effective amount of the anti-CD3 antibody according to any one of claims 1-6 and 15-31. The anti-CD3 antibody or antibody fragment as described in any one of claims 1-6 and 15-31, the bispecific binding molecule as described in any one of claims 7-10 or as in claims 11-14 Use of any of the cytotoxic active CD3 specific binding constructs in the manufacture of drugs for use in cells expressing a high number of folate receptors. A pharmaceutical Fab composition comprising a therapeutically effective amount of the anti-CD3 antibody according to any one of claims 1-6 and 15-31, the bispecific binding molecule according to any one of claims 7-10, or The cytotoxic active CD3 specific binding construct according to any one of claims 11-14 and a pharmaceutically acceptable carrier or excipient.

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