阅读说明：本技术 用于减少双特异性抗体的聚集的方法 (Methods for reducing aggregation of bispecific antibodies ) 是由 B·贾甘纳坦 J·许 M·特罗伊海特 D·单 于 2019-09-27 设计创作，主要内容包括：本文披露的本发明涉及通过在解冻后将抗体保持在某些温度下用于减少由在冷冻条件下储存而产生的双特异性抗体的聚集,例如减少双特异性T细胞接合(BiTE)抗体构建体的聚集的方法。(The invention disclosed herein relates to methods for reducing aggregation of bispecific antibodies, such as reducing aggregation of bispecific T cell junction (BiTE) antibody constructs, resulting from storage under freezing conditions by maintaining the antibodies at certain temperatures after thawing.)

1. A method of reducing aggregates of a bispecific antibody, the method comprising:

the thawed bispecific antibody is maintained at a temperature of 5 ℃ to 45 ℃ for at least 4 hours, wherein the bispecific antibody has been stored at a temperature of-20 ℃ to-40 ℃ prior to thawing.

2. The method of claim 1, wherein the bispecific antibody is a drug substance.

3. The method of claim 1 or 2, wherein the bispecific antibody is held at the temperature for a period of 4 hours to 96 hours.

4. The method of any one of claims 1-3, wherein the bispecific antibody is maintained at a temperature of 10 ℃ to 30 ℃ for a period of 8 hours to 48 hours.

5. The method of any one of claims 1-4, wherein the bispecific antibody is thawed at a temperature of 5 ℃ to 45 ℃.

6. The method of any one of claims 1-5, wherein the bispecific antibody has been stored at a temperature of-20 ℃ to-35 ℃.

7. The method of any one of claims 1-6, wherein the bispecific antibody has been stored at about-30 ℃.

8. The method of any one of claims 1-7, wherein the aggregates comprise High Molecular Weight (HMW) aggregates.

9. The method of any one of claims 1-8, wherein after the retention period, the bispecific antibody comprises less than about 1% HMW aggregates.

10. The method of claim 9, wherein the bispecific antibody comprises less than about 0.5% HMW aggregates.

11. The method of any one of claims 1-10, wherein the HMW aggregates comprise dimers of the bispecific antibody.

12. A method for making a composition comprising a bispecific antibody, the method comprising

Thawing a drug substance comprising a bispecific antibody, which has been stored at a temperature of-20 ℃ to-40 ℃, and

the thawed drug substance comprising the bispecific antibody is maintained at a temperature of 5 ℃ to 45 ℃ for at least 4 hours.

13. The method of claim 12, wherein the composition is a pharmaceutical composition comprising the bispecific antibody.

14. The method of claim 12 or 13, further comprising filtering the drug substance.

15. The method of any one of claims 12-14, further comprising aliquoting the composition into a pharmaceutical product form.

16. The method of any one of claims 12-15, wherein the drug substance is held at the temperature for a period of 4 hours to 96 hours.

17. The method of any one of claims 12-16, wherein the drug substance is maintained at a temperature of 10 ℃ to 30 ℃ for a period of 8 hours to 48 hours.

18. The method of any one of claims 12-17, wherein the drug substance is thawed at a temperature of 5 ℃ to 45 ℃.

19. The method of any one of claims 12-18, wherein the drug substance has been stored at a temperature of-20 ℃ to-35 ℃.

20. The method of any one of claims 12-19, wherein the drug substance has been stored at about-30 ℃.

21. A process for preparing a composition comprising a bispecific antibody, the process comprising holding a thawed bispecific antibody-comprising drug substance at a temperature of from 5 ℃ to 45 ℃ for at least 4 hours, wherein prior to thawing the drug substance has been frozen at a temperature of the glass transition temperature (Tg') or higher of the drug substance.

22. The method of claim 21, wherein prior to thawing, the drug substance has been frozen at a temperature of-10 ℃ to equal to or above the Tg' of the drug substance.

23. The method of claim 21 or 22, wherein the drug substance has been frozen at about-32 ℃.

24. The method of any one of claims 21-23, wherein the drug substance is held at the temperature for a period of 4 hours to 96 hours.

25. The method of any one of claims 21-24, wherein the drug substance is thawed at a temperature of 5 ℃ to 45 ℃.

26. The method of any one of claims 21-25, wherein the drug substance is maintained at the same temperature at which the drug substance thaws.

27. The method of claim 26, wherein the drug substance is thawed at a temperature of 15 ℃ to 30 ℃ and held for a period of 30 hours to 50 hours.

28. The method of any one of claims 21-27, further comprising aliquoting the composition into a pharmaceutical product form.

29. The method of any of claims 12-28, wherein after the retention period, the drug substance comprises less than about 1% HMW aggregates.

30. The method of claim 29, wherein the drug substance comprises less than about 0.5% HMW aggregates.

31. The method of claim 29 or 30, wherein the HMW aggregates comprise dimers of the bispecific antibody.

32. The method of any one of claims 1-31, wherein the drug substance comprises the bispecific antibody at a concentration of about 0.05mg/mL to about 20 mg/mL.

33. The method of any one of claims 12-32, further comprising lyophilizing the composition.

34. The method of any one of claims 12-32, further comprising spray drying the composition.

35. The method of any one of claims 1-34, wherein the bispecific antibody comprises

A first binding domain that binds to a target cell surface antigen, and

a second binding domain that binds to human CD3, and wherein the bispecific antibody is in the form of a (scFv)₂Form (a).

36. The method of claim 35, wherein the target cell surface antigen is CD19, CD33, or BCMA.

37. The method of claim 36, wherein the first binding domain comprises a VH region and a VL region, wherein:

the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO: 78;

the VH comprises the amino acid sequence of SEQ ID NO 28 and the VL comprises the amino acid sequence of SEQ ID NO 32 or 33; or

The VH comprises the amino acid sequence of SEQ ID NO:132 and the VL comprises the amino acid sequence of SEQ ID NO: 133.

38. The method of claim 37, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO 17, 40 or 135.

39. The method of any one of claims 1-38, wherein the bispecific antibody further comprises a third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain, and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker.

40. The method of claim 39, wherein the third domain comprises, in amino to carboxy order, hinge-CH 2-CH 3-linker-hinge-CH 2-CH 3.

41. The method of claim 40, wherein the third domain is an HLE domain.

42. The method of any one of claims 39-41, wherein the first binding domain binds to at least one target cell surface antigen selected from the group consisting of: CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA, or PSMA.

43. The method of claim 42, wherein the first binding domain comprises a VH region and a VL region, wherein

The VH comprises the amino acid sequence of SEQ ID NO 108 and the VL comprises the amino acid sequence of SEQ ID NO 109;

the VH comprises the amino acid sequence of SEQ ID NO 27 and the VL comprises the amino acid sequence of SEQ ID NO 32;

the VH comprises the amino acid sequence of SEQ ID NO 48 and the VL comprises the amino acid sequence of SEQ ID NO 49;

the VH comprises the amino acid sequence of SEQ ID NO 59 and the VL comprises the amino acid sequence of SEQ ID NO 60;

the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO: 78;

the VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO: 112;

the VH comprises the amino acid sequence of SEQ ID NO. 89 and the VL comprises the amino acid sequence of SEQ ID NO. 90;

the VH comprises the amino acid sequence of SEQ ID NO 100 and the VL comprises the amino acid sequence of SEQ ID NO 101;

the VH comprises the amino acid sequence of SEQ ID NO. 121 and the VL comprises the amino acid sequence of SEQ ID NO. 122;

the VH comprises the amino acid sequence of SEQ ID NO:188 and the VL comprises the amino acid sequence of SEQ ID NO: 189;

the VH comprises the amino acid sequence of SEQ ID NO:132 and the VL comprises the amino acid sequence of SEQ ID NO: 133; or

The VH comprises the amino acid sequence of SEQ ID NO 173 and the VL comprises the amino acid sequence of SEQ ID NO 174.

44. The method of claim 43, wherein the bispecific antibody comprises the amino acid sequence of SEQ ID NO 63, 114, 41, 82, 136, 104, 93, 177, 125, 190 or 52.

45. The method of claim 44, wherein the bispecific antibody consists of the amino acid sequence of SEQ ID NO 63, 114, 41, 82, 136, 104, 93, 177, 125, 190 or 52.

46. The method of any one of claims 1-34, wherein the bispecific antibody is a bispecific masked antigen binding protein.

47. The method of claim 46, wherein the bispecific masked antigen binding protein comprises

(a) A first antibody or antigen-binding fragment thereof (AB1) that binds to a first antigen, and a masking domain (MD1) coupled to AB1, wherein the MD1 comprises

(1) A first masking peptide (MP1) that inhibits or reduces the binding of AB1 to its antigen, and

(2) a protein recognition site (PR1), wherein binding of a protein or protease to the PR1 or cleavage of the PR1 increases binding of AB1 to its antigen;

(b) a second antibody or antigen-binding fragment thereof (AB2) that binds a second antigen, and a second masking domain (MD2) coupled to AB2, wherein the MD2 comprises:

(1) a second Masking Peptide (MPs) that inhibits or reduces the binding of AB2 to its antigen, and

(2) a second protein recognition site (PR2), wherein binding of a protein or protease to the PR2 or cleavage of the PR2 increases binding of AB2 to its antigen.

48. The method of claim 46 or 47, wherein the PR1 and PR2 comprise the same protein recognition sequence.

49. The method of any one of claims 46-48, wherein the AB1 binds to human CD3 and the AB2 binds to human EGFR.

Technical Field

The present application relates to methods for reducing aggregation of bispecific antibodies. More specifically, the present application relates to methods for reducing aggregation of bispecific antibodies (e.g., bispecific T cell-engaging antibodies) disclosed herein resulting from storage under refrigerated conditions.

Background

Therapeutic proteins such as antibodies are an important class of drugs directed to patients. Typically, therapeutic proteins are produced in eukaryotic cells and purified in large quantities. Since proteins are sensitive to temperature changes, bulk drugs of these therapeutic proteins are stored and transported under refrigerated conditions at temperatures ranging from-20 ℃ to-80 ℃. Freezing extends the shelf life of the therapeutic protein and provides flexibility in scheduling the final formulation and filling of the protein into commercial product packaging. Operating infrastructure and logistics for storage and transportation are advantageous at higher temperatures in this range.

The freeze/thaw process may be a source of stress for the protein. For example, water crystallizes during the freezing of a protein drug substance, which may result in a concentration level of the protein molecules that is several times the initial level. Changes in concentration may result in a loss of thermodynamic stability of the protein, leading to folding events and resulting aggregation. See, for example, 18Lam Philippe et al, Quality by Design for Biopharmaceutical Drug Product Development Design Quality, page 159-189 (edited by Jameel F. et al, 2015). Similar stresses may be involved in the thawing process, and they may be exacerbated by the higher temperatures required to melt the ice in a timely manner. As above. Thus, when a drug substance comprising a therapeutic protein is stored and/or transported under freezing conditions, protein aggregates (e.g., High Molecular Weight (HMW) aggregates) may be produced as a result of the freeze/thaw process. However, the presence of aggregates in the drug substance is undesirable as aggregates may negatively affect the stability, immunogenicity and efficacy of the protein. There is a need for methods for reducing protein aggregation resulting from storage under refrigerated conditions.

Disclosure of Invention

Provided herein are methods for reducing aggregation of bispecific antibodies, particularly bispecific T cell-engaging antibodies (bites), resulting from storage under refrigerated conditions. The present application relates to the surprising discovery that maintaining a bispecific antibody at certain temperatures for a period of time after thawing reduces aggregates formed upon storage under freezing conditions.

In one embodiment, disclosed herein is a method for reducing aggregates of a bispecific antibody, comprising maintaining a thawed bispecific antibody at a temperature of about 5 ℃ to about 45 ℃ for at least 4 hours, wherein prior to thawing, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃. In one embodiment, the thawed bispecific antibody is maintained at the temperature for a period of about 4 hours to about 96 hours. In one embodiment, the thawed bispecific antibody is maintained at a temperature of about 10 ℃ to about 30 ℃ for a period of about 10 hours to about 48 hours. In one embodiment, the thawed bispecific antibody is maintained at a temperature of about 10 ℃ to about 30 ℃ for a period of 8 hours to 48 hours.

In one embodiment, the bispecific antibody is thawed at a temperature of about 5 ℃ to about 45 ℃. In one embodiment, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-35 ℃ prior to thawing, and in another embodiment, the bispecific antibody has been stored at about-30 ℃ prior to thawing. In one embodiment, the aggregates comprise High Molecular Weight (HMW) aggregates. In one embodiment, the HMW aggregate comprises a dimer of a bispecific antibody. In one embodiment, the bispecific antibody comprises less than about 1% HMW aggregates after the retention period, in another embodiment, the bispecific antibody comprises less than about 0.5% HMW aggregates after the retention period.

In one embodiment, disclosed herein is a method for preparing a composition comprising a bispecific antibody, the method comprising thawing a drug substance comprising a bispecific antibody that has been stored at a temperature of about-20 ℃ to about-40 ℃, and holding the thawed drug substance comprising a bispecific antibody at a temperature of about 5 ℃ to about 45 ℃ for at least 4 hours. In one embodiment, the drug substance is held at this temperature for a period of time of about 4 hours to about 96 hours. In another embodiment, the drug substance is held at a temperature of about 10 ℃ to about 30 ℃ for a period of about 10 hours to about 48 hours. In another embodiment, the drug substance is held at a temperature of about 10 ℃ to about 30 ℃ for a period of 8 hours to 48 hours. In one embodiment, the drug substance is thawed at a temperature of about 5 ℃ to about 45 ℃. In one embodiment, the drug substance has been stored at a temperature of about-20 ℃ to about-35 ℃, in another embodiment, the drug substance has been stored at about-30 ℃. In one embodiment, the method for preparing a composition further comprises filtering the drug substance, and in another embodiment, the method for preparing a composition further comprises aliquoting the composition into a pharmaceutical product form. In one embodiment, the composition is a pharmaceutical composition comprising a bispecific antibody.

In one embodiment, disclosed herein is a method for preparing a composition comprising a bispecific antibody, the method comprising holding a thawed drug substance comprising a bispecific antibody at a temperature of about 5 ℃ to about 45 ℃ for at least 4 hours, wherein prior to thawing the drug substance has been frozen at the glass transition temperature (Tg') or higher of the drug substance. In one embodiment, the drug substance has been frozen at a temperature of about-10 ℃ to a temperature equal to or above the glass transition temperature of the drug substance, and in another embodiment, the drug substance has been frozen at about-32 ℃. In one embodiment, the drug substance is held at this temperature for a period of time of about 4 hours to about 96 hours. In one embodiment, the drug substance is thawed at a temperature of about 5 ℃ to about 45 ℃. In another embodiment, the bulk drug is maintained at the same temperature at which the bulk drug is thawed. In another embodiment, the drug substance is thawed at the same temperature of about 15 ℃ to about 30 ℃ and held for a period of 30 hours to 50 hours. In another embodiment, the method for preparing a composition further comprises aliquoting the composition into a pharmaceutical product form. In another embodiment, the method for preparing a composition further comprises lyophilizing the composition. In yet another embodiment, the method for preparing a composition further comprises spray drying the composition.

In one embodiment, the drug substance comprises less than about 1% HMW aggregates after the retention period, and in another embodiment, the drug substance comprises less than about 0.5% HMW aggregates. In one embodiment, the HMW aggregate comprises a dimer of a bispecific antibody.

In one embodiment, the drug substance comprises a bispecific antibody at a concentration of about 0.05mg/mL to about 20 mg/mL.

In one embodiment, the bispecific antibody is a bispecific T cell-engaging antibody (BiTE). In one embodiment, the bispecific antibody comprises a first binding domain that binds to a target cell surface antigen, and a second binding domain that binds to human CD3, and wherein the bispecific antibody is in the form of (scFv) 2. In one embodiment, the bispecific antibody comprises a first binding domain that binds to a target cell surface antigen selected from CD19, CD33, or BCMA, and a second binding domain that binds to human CD3, and wherein the bispecific antibody is in the form of (scFv) 2.

In one embodiment, the bispecific antibody comprises a first binding domain and a second binding domain, wherein the first binding domain comprises a VH region and a VL region, and wherein: the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO: 78; or the VH comprises the amino acid sequence of SEQ ID NO 28 and the VL comprises the amino acid sequence of SEQ ID NO 32 or 33; or the VH comprises the amino acid sequence of SEQ ID NO:132 and the VL comprises the amino acid sequence of SEQ ID NO: 133. In another embodiment, the bispecific antibody comprises the amino acid sequence of SEQ ID NO 17, 40 or 135.

In one embodiment, the bispecific antibody is a BiTE, wherein the BiTE further comprises a third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain, and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker. In another embodiment, the third domain comprises, in amino to carboxyl order, the hinge-CH 2-CH 3-linker-hinge-CH 2-CH 3. In one embodiment, the third domain is a half-life extended (HLE) domain.

In one embodiment, the bispecific antibody comprises a first binding domain, a second binding domain, and a third domain, wherein the first binding domain binds to at least one target cell surface antigen selected from the group consisting of: CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA, or PSMA, the second binding domain binding to human CD3, and the third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain, and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker.

In one embodiment, the bispecific antibody comprises a first binding domain comprising a VH region and a VL region, and binds to at least one target cell surface antigen selected from the group consisting of: CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA or PSMA, the second binding domain binding to human CD3, and the third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker, and wherein (a) the VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO: 109; or (b) the VH comprises the amino acid sequence of SEQ ID NO:27 and the VL comprises the amino acid sequence of SEQ ID NO: 32; or (c) the VH comprises the amino acid sequence of SEQ ID NO:48 and the VL comprises the amino acid sequence of SEQ ID NO: 49; or (d) the VH comprises the amino acid sequence of SEQ ID NO:59 and the VL comprises the amino acid sequence of SEQ ID NO: 60; or (e) the VH comprises the amino acid sequence of SEQ ID NO:77 and the VL comprises the amino acid sequence of SEQ ID NO: 78; or (f) the VH comprises the amino acid sequence of SEQ ID NO:108 and the VL comprises the amino acid sequence of SEQ ID NO: 112; or (g) the VH comprises the amino acid sequence of SEQ ID NO:89 and the VL comprises the amino acid sequence of SEQ ID NO: 90; or (h) the VH comprises the amino acid sequence of SEQ ID NO:100 and the VL comprises the amino acid sequence of SEQ ID NO: 101; or (i) the VH comprises the amino acid sequence of SEQ ID NO:121 and the VL comprises the amino acid sequence of SEQ ID NO: 122; or (j) the VH comprises the amino acid sequence of SEQ ID NO:188 and the VL comprises the amino acid sequence of SEQ ID NO: 189; or (k) the VH comprises the amino acid sequence of SEQ ID NO:132 and the VL comprises the amino acid sequence of SEQ ID NO: 133; or (l) the VH comprises the amino acid sequence of SEQ ID NO:173 and the VL comprises the amino acid sequence of SEQ ID NO: 174.

In one embodiment, the bispecific antibody comprises a first binding domain that binds to at least one target cell surface antigen selected from the group consisting of: CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, CD70, BCMA or PSMA, the second binding domain binding to human CD3 and the third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain and a CH3 domain, wherein the two polypeptide monomers are linked to each other via a peptide linker, and wherein the bispecific antibody comprises or consists of an amino acid sequence selected from SEQ ID NOs 63, 114, 41, 82, 136, 104, 93, 177, 125, 190 or 52.

In one embodiment, the bispecific antibody is a bispecific masked antigen binding protein. In one embodiment, the bispecific masked antigen binding protein comprises (a) a first antibody or antigen binding fragment thereof (AB1) that binds to a first antigen, and a masking domain (MD1) coupled to AB1, wherein the MD1 comprises (1) a first masking peptide (MP1) that inhibits or reduces the binding of AB1 to its antigen, and (2) a protein recognition site (PR1), wherein binding of a protein or protease to the PR1 or cleavage of the PR1 increases the binding of AB1 to its antigen; (b) a second antibody or antigen-binding fragment thereof (AB2) that binds a second antigen, and a second masking domain (MD2) coupled to AB2, wherein the MD2 comprises: (1) second Masking Peptides (MPs) that inhibit or reduce binding of AB2 to its antigen, and (2) a second protein recognition site (PR2), wherein binding of a protein or protease to the PR2 or cleavage of the PR2 increases binding of AB2 to its antigen. In one embodiment, PR1 and PR2 comprise the same protein recognition sequence. In another embodiment, AB1 binds to human CD3 and AB2 binds to human EGFR.

Drawings

FIG. 1 shows the increase in aggregate levels (. DELTA.HMW%) after storage of various HLE BITE molecules at-20 ℃ for one month.

Figure 2A shows the increase in aggregate levels (Δ HMW%) of DLL3xCD3 HLE BiTE after one month of storage at-20 ℃ in compositions with different pH.

Figure 2B shows the increase in aggregate levels (Δ HMW%) of DLL3xCD3 HLE BiTE after one month of storage at different temperatures.

FIGS. 3A, 3B, 3C, and 3D show increased levels of HMW aggregates in various HLE BiTEs after storage at-20 ℃ or-30 ℃ and decreased levels of aggregates after thawing after 24 hours at room temperature.

FIG. 4 shows the decrease in HMW levels of a BiTE molecule as a function of retention time and retention temperature.

Figure 5 shows the stabilization of benzyl alcohol.

Detailed Description

Method

Described herein are methods of reducing aggregates of bispecific antibodies, particularly aggregates formed when bispecific antibodies are stored under refrigerated conditions. As used herein, the term "aggregate" or "aggregation" refers to the association of two or more molecules. In certain embodiments, the aggregates are High Molecular Weight (HMW) aggregates having a molecular weight and/or size greater than that of the non-aggregated molecules. In certain embodiments, the aggregate comprises an association of two or more antibody molecules. In certain embodiments, the aggregate comprises an association of two or more bispecific antibody molecules, including dimers of bispecific antibodies. In certain embodiments, the bispecific antibody is a bispecific T cell-engaging antibody (BiTE).

The presence and/or level of aggregates can be determined by techniques known in the art, such as techniques for determining molecular size, e.g., size exclusion chromatography, cation exchange chromatography, X-ray diffraction, modulated differential scanning calorimetry (mDSC), and native polyacrylamide gel electrophoresis (PAGE). In one embodiment, the presence and/or level of protein aggregates is determined by size exclusion HPLC (SE-HPLC). In another embodiment, the presence and/or level of protein aggregates is determined by size exclusion ultra-HPLC (SE-UHPLC).

In one aspect, disclosed herein is a method for reducing aggregation of a bispecific antibody, the method comprising maintaining a thawed bispecific antibody at a temperature for at least 4 hours, wherein the bispecific antibody has been stored under freezing conditions prior to thawing. As used herein, the term "thawed bispecific antibody" or "thawed bispecific antibody-containing drug substance" is understood to refer to a liquid bispecific antibody or bispecific antibody-containing liquid drug substance from a frozen state but produced as a result of exposure to warmth. In certain embodiments, the thawed bispecific antibody or thawed bispecific antibody-containing drug substance is a liquid bispecific antibody or bispecific antibody-containing drug substance that is free or substantially free of frozen material.

In certain embodiments, the bispecific antibody is a drug substance. As used herein, the term "drug substance" is understood to refer to a recombinant protein (e.g., bispecific antibody) that has been sufficiently purified or isolated from contaminating proteins, lipids, and nucleic acids (e.g., present in a liquid culture medium or from a host cell (e.g., from a mammalian, yeast, or bacterial host cell)) and biological contaminants (e.g., viral and bacterial contaminants) and that can be formulated into a pharmaceutical composition without further substantial purification and/or decontamination steps. The term drug substance encompasses compositions comprising a substantially purified recombinant protein (e.g., a bispecific antibody) and one or more pharmaceutically suitable excipients.

In certain embodiments, the thawed drug substance is a composition comprising one or more excipients in addition to a recombinant protein (e.g., a bispecific antibody). Excipients suitable for use in pharmaceutical compositions may be used. Exemplary excipients include buffers (e.g., acetate buffer, glutamate buffer, citrate buffer, lactic buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, or phosphate buffer), sugars (e.g., glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, or xylitol), and surfactants (e.g., polysorbate 20 or polysorbate 80). In certain embodiments, the thawed drug substance is a composition comprising a recombinant protein (e.g., a bispecific antibody), a buffer (e.g., a glutamate buffer or a citrate buffer), a saccharide (e.g., sucrose), and optionally a surfactant (e.g., polysorbate 80). The pH of the thawed drug substance can be in the range of about 3.0 to 7.0 or about 4.0 to about 6.0.

In certain embodiments, the frozen bispecific antibody is thawed by exposure to elevated temperature. In certain embodiments, the bispecific antibody is thawed at a temperature of from about 0 ℃ to about 50 ℃, or from about 0 ℃ to about 40 ℃, or from about 0 ℃ to about 30 ℃, or from about 5 ℃ to about 45 ℃, or from about 5 ℃ to about 30 ℃, or from about 10 ℃ to about 50 ℃, or from about 10 ℃ to about 40 ℃, or from about 10 ℃ to about 30 ℃, or from about 15 ℃ to about 50 ℃, or from about 15 ℃ to about 40 ℃, or from about 15 ℃ to about 30 ℃, or from about 20 ℃ to about 30 ℃, or from about 25 ℃ to about 30 ℃. In certain embodiments, the bispecific antibody is thawed at a temperature of about 0 ℃ to about 25 ℃, or about 5 ℃ to about 25 ℃, or about 10 ℃ to about 25 ℃, or about 15 ℃ to about 25 ℃, or about 20 ℃ to about 25 ℃. In certain embodiments, the bispecific antibody is thawed at a temperature of about 0 ℃, about 5 ℃, about 10 ℃, about 15 ℃, about 20 ℃, about 25 ℃, or about 30 ℃, or about 40 ℃, or about 45 ℃, or about 50 ℃. As will be appreciated by one of ordinary skill in the art, the bispecific antibody may be gently mixed during thawing to ensure a uniform distribution of temperature and/or disrupt the concentration gradient formed during thawing. Gentle mixing can be achieved, for example, by using an inclined shaker or gently inverting the container of bispecific antibody. Alternatively, the bispecific antibody may be gently mixed after thawing.

In certain embodiments, the method comprises holding the thawed bispecific antibody at a temperature for at least 4 hours. In certain embodiments, the holding temperature is from about 0 ℃ to about 50 ℃, or from about 0 ℃ to about 40 ℃, or from about 5 ℃ to about 50 ℃, or from about 5 ℃ to about 45 ℃, or from about 5 ℃ to about 40 ℃, or from about 5 ℃ to about 30 ℃, or from about 10 ℃ to about 50 ℃, or from about 10 ℃ to about 45 ℃, or from about 10 ℃ to about 40 ℃, or from about 10 ℃ to about 30 ℃, or from about 15 ℃ to about 40 ℃, or from about 15 ℃ to about 30 ℃. In certain embodiments, the holding temperature is about 15 ℃ to about 25 ℃. In certain embodiments, the holding temperature is about 5 ℃, or about 10 ℃, or about 15 ℃, or about 17 ℃, or about 19 ℃, or about 20 ℃, or about 23 ℃, or about 25 ℃, or about 27 ℃, or about 30 ℃, or about 35 ℃, or about 40 ℃, or about 45 ℃. In certain embodiments, the thawed bispecific antibody is maintained at any one of the above temperatures for about 4 hours to about 120 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours, or about 10 hours to about 120 hours, or about 10 hours to about 96 hours, or about 10 hours to about 72 hours, or about 10 hours to about 48 hours, or about 10 hours to about 24 hours, or a period of time from about 24 hours to about 120 hours, or from about 24 hours to about 100 hours, or from about 24 hours to about 96 hours, or from about 24 hours to about 72 hours, or from about 24 hours to about 48 hours, or from about 48 hours to about 100 hours, or from about 48 hours to about 96 hours, or from about 48 hours to about 72 hours, or from about 72 hours to about 100 hours, or from about 72 hours to about 96 hours.

In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 5 ℃ to about 45 ℃ for a period of about 4 hours to about 120 hours, or a period of about 4 hours to about 100 hours, or a period of about 4 hours to about 96 hours, or a period of about 4 hours to about 72 hours, or a period of about 4 hours to about 48 hours, or a period of about 4 hours to about 24 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 10 ℃ to about 30 ℃ for a period of about 4 hours to about 24 hours, or a period of about 10 hours to about 120 hours, or a period of about 10 hours to about 96 hours, or a period of about 10 hours to about 72 hours, or a period of about 10 hours to about 48 hours, or a period of about 10 hours to about 24 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 30 ℃ to about 45 ℃ for a period of about 4 hours to about 24 hours or a period of about 4 hours to about 10 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 15 ℃ to about 45 ℃ for a period of about 4 hours to about 120 hours, or a period of about 4 hours to about 50 hours, or a period of about 4 hours to about 24 hours, or a period of about 8 hours to about 50 hours, or a period of about 15 hours to about 50 hours, or a period of about 4 hours to about 100 hours, or a period of about 8 hours to about 100 hours, or a period of about 15 hours to about 100 hours, or a period of about 24 hours to about 96 hours, or a period of about 24 hours to about 72 hours, or a period of about 24 hours to about 48 hours, or a period of about 48 hours to about 100 hours, or a period of about 48 hours to about 96 hours, or a period of about 48 hours to about 72 hours, or a period of about 72 hours to about 100 hours, or a period of about 72 hours to about 96 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 15 ℃ to about 30 ℃ for a period of about 4 hours to about 24 hours, or a period of about 10 hours to about 120 hours, or a period of about 10 hours to about 96 hours, or a period of about 10 hours to about 72 hours, or a period of about 10 hours to about 48 hours, or a period of about 10 hours to about 24 hours, or a period of about 24 hours to about 96 hours, or a period of about 24 hours to about 72 hours, or a period of about 24 hours to about 48 hours, or a period of about 48 hours to about 100 hours, or a period of about 48 hours to about 96 hours, or a period of about 48 hours to about 72 hours, or a period of about 72 hours to about 100 hours, or a period of about 72 hours to about 96 hours. In certain embodiments, the thawed bispecific antibody is maintained at a temperature of about 15 ℃ to about 25 ℃ for a period of about 4 hours to about 24 hours, or a period of about 10 hours to about 48 hours, or a period of about 15 hours to about 30 hours, or a period of about 15 hours to about 24 hours, or a period of about 24 hours to about 120 hours, or a period of about 24 hours to about 96 hours, or a period of about 24 hours to about 72 hours, or a period of about 24 hours to about 48 hours, or a period of about 48 hours to about 100 hours, or a period of about 48 hours to about 96 hours, or a period of about 48 hours to about 72 hours, or a period of about 72 hours to about 100 hours, or a period of about 72 hours to about 96 hours. In another embodiment, the thawed bispecific antibody is maintained at a temperature of about 15 ℃ to about 30 ℃ for a period of about 10 hours, or about 15 hours, or about 20 hours, or about 24 hours, or about 36 hours, or about 48 hours, or about 60 hours, or about 72 hours, or about 84 hours, or about 96 hours, or about 120 hours.

Before thawing, the bispecific antibody has been stored under frozen conditions. In certain embodiments, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-50 ℃. In certain embodiments, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃. In certain embodiments, the bispecific antibody has been stored at a temperature of about-25 ℃ to about-35 ℃. In certain embodiments, the bispecific antibody has been stored at a temperature of about-20 ℃, about-30 ℃, about-35 ℃, about-40 ℃, or about-50 ℃. In certain embodiments, the bispecific antibody has been stored at a temperature of about-30 ℃. In certain embodiments, the bispecific antibody has been stored at a temperature of 0 ℃ to a temperature equal to or above the glass transition temperature (Tg ') of the antibody or Tg' of a composition comprising the antibody. In certain embodiments, the bispecific antibody has been stored at a temperature of about-10 ℃ to equal to or above the Tg 'of the antibody or the Tg' of a composition comprising the antibody.

In certain embodiments, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃ for a period of about one day to about 5 years. In certain embodiments, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃ for a period of about one week to about 5 years or about one month to about 5 years. In certain embodiments, the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃ for a period of about one week, about two weeks, about three weeks, about four weeks, about one month, about six months, about eighteen months, about one year, about two years, about three years, about four years, or about 5 years.

As used herein, the term "about," when used to modify a particular value or range, is understood to mean that there can be variations within the given value or range, including greater than or less than 20% (e.g., 10%, 5%, 4%, 3%, 2%, or 1%) of the stated value or range.

As used herein, the term "storage" or "stored" refers to the placement or retention of the bispecific antibody under certain conditions for later use or further processing. In certain embodiments, the antibody is placed at any of the temperatures described above, which can be achieved by using, for example, a freezer, refrigerated truck, or transportation device that can maintain the desired temperature. As will be appreciated by one of ordinary skill in the art, the antibody is frozen when stored at the storage temperatures described above.

The bispecific antibody can be stored in any suitable container that can maintain its integrity at storage temperatures. Exemplary containers include vials, bottles, bags, and large glass bottles. Such containers are well known in the art and are commercially available. In certain embodiments, the bispecific antibody is stored in a disposable container, such as a commercially available flexible freeze thaw container, e.g.,an FFT system. The volume of bispecific antibody under storage is determined by the volume of the container used for storage. In certain embodiments, the volume of the container is 5mL, 10mL, 100mL, 500mL, 1 liter (L), 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, or 12L. In certain embodiments, the volume of bispecific antibody under storage is about 5mL, about 10mL, about 100mL,about 500mL, about 1 liter (L), about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, or about 12L.

In certain embodiments, the concentration of bispecific antibody under storage ranges from about 0.01mg/mL to about 25mg/mL, or from about 0.05mg/mL to about 25mg/mL, or from about 0.1mg/mL to about 25mg/mL, or from about 0.5mg/mL to about 25mg/mL, or from about 1mg/mL to about 25 mg/mL. In certain embodiments, the concentration of bispecific antibody under storage ranges from about 1mg/mL to about 20mg/mL, or from about 1mg/mL to about 15mg/mL, or from about 1mg/mL to about 10mg/mL, or from about 1mg/mL to about 5 mg/mL. In certain embodiments, the concentration of bispecific antibody under storage is about 0.01mg/mL, about 0.05mg/mL, about 0.1mg/mL, about 0.5mg/mL, about 1mg/mL, about 2mg/mL, about 3mg/mL, about 4mg/mL, about 5mg/mL, about 6mg/mL, about 7mg/mL, about 8mg/mL, about 9mg/mL, about 10mg/mL, about 11mg/mL, about 12mg/mL, about 13mg/mL, about 14mg/mL, about 15mg/mL, about 16mg/mL, about 17mg/mL, about 18mg/mL, about 19mg/mL, about 20mg/mL, or about 25 mg/mL.

Also disclosed herein are methods for making compositions comprising bispecific antibodies. In certain embodiments, the method comprises thawing a drug substance comprising the bispecific antibody that has been stored under frozen conditions, and holding the thawed drug substance at a temperature for at least 4 hours.

In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-20 ℃ to about-50 ℃. In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-20 ℃ to about-35 ℃. In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-25 ℃ to about-35 ℃. In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-20 ℃, about-30 ℃, about-35 ℃, about-40 ℃, or about-50 ℃. In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-30 ℃.

In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-20 ℃ to about-50 ℃ for a period of about one day to about 5 years. In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃ for a period of about one week to about 5 years or about one month to about 5 years. In certain embodiments, the drug substance comprising the bispecific antibody has been stored at a temperature of about-20 ℃ to about-40 ℃ for a period of about one week, about two weeks, about three weeks, about four weeks, about one month, about six months, about eighteen months, about one year, about two years, about three years, about four years, or about 5 years.

In certain embodiments, a method for making a composition comprising a bispecific antibody comprises holding a thawed drug substance comprising a bispecific antibody at a temperature wherein, prior to thawing, the drug substance has been frozen at a temperature at or above the glass transition temperature (Tg') of the drug substance for at least 4 hours. In certain embodiments, the drug substance comprising the bispecific antibody has been frozen at a temperature of about-10 ℃ to equal to or above the Tg' of the drug substance. In certain embodiments, the drug substance comprising the bispecific antibody has been frozen at a temperature of about-20 ℃ to equal to or above the Tg' of the drug substance. In certain embodiments, the drug substance comprising the bispecific antibody has been frozen at a temperature equal to or above the Tg 'of the drug substance (e.g., about-10 ℃ to equal to or above Tg') for a period of about one day to about 5 years, or about one week to about 5 years. In certain embodiments, the drug substance comprising the bispecific antibody has been frozen at a temperature equal to or above the Tg 'of the drug substance (e.g., about-10 ℃ to equal to or above Tg') for a period of about one month, about six months, about one year, about eighteen months, about 2 years, about three years, about 4 years, or about 5 years. In certain embodiments, the Tg' of the drug substance is about-30 deg.C, or about-32 deg.C, or about-35 deg.C. In certain embodiments, the drug substance comprising the bispecific antibody has been frozen at a temperature of about-32 ℃ for a period of about one month, about six months, about one year, about eighteen months, about 2 years, about three years, about 4 years, or about 5 years.

As will be appreciated by one of ordinary skill in the art, the glass transition temperature of a bispecific antibody or drug substance comprising a bispecific antibody can be determined by methods known in the art, such as Differential Scanning Calorimetry (DSC), thermomechanical analysis (TMA), Dynamic Mechanical Analysis (DMA), and dilatometry.

The drug substance comprising the bispecific antibody may have been stored or frozen in any suitable container that can maintain its integrity at storage/freezing temperatures. Exemplary containers include vials, bottles, bags, and large glass bottles. Such containers are well known in the art and are commercially available. In certain embodiments, the drug substance comprising the bispecific antibody is stored in a disposable container, such as a commercially available flexible freeze thaw container, e.g.,an FFT system. The volume of drug substance comprising bispecific antibody under storage is determined by the volume of the container used for storage. In certain embodiments, the volume of the container is 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, or 12L.

Drug substances comprising bispecific antibodies can be thawed by exposure to elevated temperatures. In certain embodiments, the drug substance is thawed at a temperature of from about 0 ℃ to about 50 ℃, or from about 0 ℃ to about 40 ℃, or from about 0 ℃ to about 30 ℃, or from about 5 ℃ to about 45 ℃, or from about 5 ℃ to about 30 ℃, or from about 10 ℃ to about 30 ℃, or from about 15 ℃ to about 30 ℃, or from about 20 ℃ to about 30 ℃, or from about 25 ℃ to about 30 ℃. In certain embodiments, the drug substance is thawed at a temperature of from about 0 ℃ to about 25 ℃, or from about 5 ℃ to about 25 ℃, or from about 10 ℃ to about 25 ℃, or from about 15 ℃ to about 25 ℃, or from about 20 ℃ to about 25 ℃. In certain embodiments, the drug substance is thawed at a temperature of about 0 ℃, about 5 ℃, about 10 ℃, about 15 ℃, about 20 ℃, about 25 ℃, about 27 ℃, or about 30 ℃, or about 40 ℃, or about 45 ℃, or about 50 ℃.

In certain embodiments, the method comprises maintaining the thawed drug substance at a temperature of from about 0 ℃ to about 50 ℃, or from about 0 ℃ to about 40 ℃, or from about 5 ℃ to about 50 ℃, or from about 5 ℃ to about 45 ℃, or from about 10 ℃ to about 50 ℃, or from about 10 ℃ to about 45 ℃, or from about 10 ℃ to about 30 ℃, or from about 15 ℃ to about 40 ℃, or from about 15 ℃ to about 30 ℃. In certain embodiments, the thawed drug substance is maintained at a temperature of about 15 ℃ to about 25 ℃. In certain embodiments, the thawed drug substance is maintained at a temperature of about 5 ℃, or about 10 ℃, or about 15 ℃, or about 17 ℃, or about 19 ℃, or about 21 ℃, or about 23 ℃, or about 25 ℃, or about 27 ℃, or about 29 ℃, or about 40 ℃, or about 45 ℃. In certain embodiments, the thawed drug substance is maintained at any of the above temperatures for a period of about 4 hours to about 150 hours, or a period of about 4 hours to about 100 hours, or a period of about 4 hours to about 72 hours, or a period of about 4 hours to about 48 hours, or a period of about 4 hours to about 24 hours, or a period of about 10 hours to about 150 hours, or a period of about 10 hours to about 100 hours, or a period of about 10 hours to about 72 hours, or a period of about 10 hours to about 48 hours, or a period of about 10 hours to about 24 hours, or a period of about 24 hours to about 150 hours, or a period of about 24 hours to about 120 hours, or a period of about 24 hours to about 100 hours, or a period of about 24 hours to about 96 hours, or a period of about 24 hours to about 72 hours, or a period of about 24 hours to about 48 hours, a, A time period of about 48 hours to about 100 hours, or a time period of about 48 hours to about 96 hours, or a time period of about 48 hours to about 72 hours, or a time period of about 72 hours to about 100 hours, or a time period of about 72 hours to about 96 hours.

In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 5 ℃ to about 45 ℃ for at least about 4 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 5 ℃ to about 45 ℃ for a period of about 4 hours to about 120 hours, or a period of about 4 hours to about 96 hours, or a period of about 4 hours to about 72 hours, or a period of about 4 hours to about 48 hours, or a period of about 4 hours to about 24 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 10 ℃ to about 45 ℃ for a period of about 4 hours to about 100 hours or a period of about 4 hours to about 50 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 15 ℃ to about 40 ℃ for a period of about 4 hours to about 150 hours, or for a period of about 4 hours to about 120 hours, or for a period of about 4 hours to about 96 hours, or for a period of about 4 hours to about 72 hours, or for a period of about 4 hours to about 48 hours, or for a period of about 4 hours to about 24 hours, or for a period of about 10 hours to about 120 hours, or for a period of about 10 hours to about 96 hours, or for a period of about 10 hours to about 72 hours, or for a period of about 10 hours to about 48 hours, or for a period of about 10 hours to about 24 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 15 ℃ to about 30 ℃ for a period of about 10 hours to about 96 hours, or a period of about 10 hours to about 72 hours, or a period of about 10 hours to about 48 hours, or a period of about 10 hours to about 24 hours, or a period of about 24 hours to about 96 hours, or a period of about 24 hours to about 72 hours, or a period of about 24 hours to about 48 hours, or a period of about 48 hours to about 100 hours, or a period of about 48 hours to about 96 hours, or a period of about 48 hours to about 72 hours, or a period of about 72 hours to about 100 hours, or a period of about 72 hours to about 96 hours. In certain embodiments, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 15 ℃ to about 25 ℃ for a period of about 15 hours to about 96 hours, or a period of about 15 hours to about 72 hours, or a period of about 15 hours to about 48 hours, or a period of about 24 hours to about 96 hours, or a period of about 24 hours to about 72 hours, or a period of about 24 hours to about 48 hours, or a period of about 48 hours to about 100 hours, or a period of about 48 hours to about 96 hours, or a period of about 48 hours to about 72 hours, or a period of about 72 hours to about 100 hours, or a period of about 72 hours to about 96 hours. In one embodiment, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 10 ℃ to about 30 ℃ for a period of about 10 hours to about 96 hours, or for a period of about 10 hours to about 72 hours, or for a period of about 10 hours to about 48 hours, or for a period of about 10 hours to about 24 hours, or for a period of about 24 hours to about 96 hours, or for a period of about 24 hours to about 72 hours, or for a period of about 24 hours to about 48 hours, or for a period of about 48 hours to about 96 hours, or for a period of about 72 hours to about 96 hours. In another embodiment, the thawed drug substance comprising the bispecific antibody is maintained at a temperature of about 10 ℃ to about 30 ℃ for a period of about 4 hours, or about 10 hours, or about 24 hours, or about 48 hours, or about 60 hours, or about 72 hours, or about 96 hours, or about 120 hours, or about 150 hours.

In certain embodiments, the thawed bispecific antibody-containing drug substance is maintained at a temperature of about 5 ℃ to about 45 ℃ for a period of time such that the level of aggregates in the bispecific antibody-containing drug substance is reduced to about the same level as before storage under frozen conditions. In certain embodiments, the thawed bispecific antibody-containing drug substance is held at a temperature of about 5 ℃ to about 45 ℃ for about 4 hours until the time at which the level of aggregates in the bispecific antibody-containing drug substance decreases to about the same level as before storage under frozen conditions. As will be appreciated by one of ordinary skill in the art, the time required for the level of aggregates in a thawed drug substance to decrease to about the same level as before storage under frozen conditions at a particular temperature can be determined by measuring the level of aggregates in the drug substance before storage under frozen conditions and at different time points after thawing using methods known and used in the art (e.g., SE-UHPLC), see, e.g., example 5.

In certain embodiments, the drug substance is maintained at the same temperature at which it is thawed. For example, a drug substance comprising a bispecific antibody can be thawed at a temperature (e.g., any of the thawing and holding temperatures disclosed above), such as a temperature of about 5 ℃ to about 45 ℃, and then, after thawing, held at the same temperature for at least 4 hours (e.g., a period of about 4 hours to about 150 hours, or about 4 hours to about 120 hours, or about 4 hours to about 96 hours, or about 4 hours to about 72 hours, or about 4 hours to about 48 hours, or about 4 hours to about 24 hours, or about 8 hours to about 96 hours, or about 8 hours to about 72 hours, or about 8 hours to about 48 hours, or about 24 hours to about 72 hours, or about 24 hours to about 48 hours). One of ordinary skill in the art can readily determine whether a drug substance has thawed.

In certain embodiments, the drug substance is maintained at the same temperature at which it is thawed and the drug substance is maintained at the same temperature for the total period of time (time to thaw and hold) until the level of aggregates in the drug substance comprising the bispecific antibody is reduced to about the same level as before storage under frozen conditions. As shown in the examples, one of ordinary skill in the art can determine the level of aggregates in the drug substance at various time points before freezing and after thawing using methods known in the art (e.g., SE-UHPLC). In one embodiment, the drug substance is thawed at the same temperature of about 5 ℃ to about 45 ℃ and held for a total period of time of about 30 hours to about 100 hours, or a total period of time of about 30 hours to about 90 hours, or a total period of time of about 30 hours to about 80 hours, or a total period of time of about 30 hours to about 70 hours, or a total period of time of about 30 hours to about 60 hours, or a total period of time of about 30 hours to about 50 hours. In one embodiment, the drug substance is thawed at the same temperature of about 15 ℃ to about 30 ℃ and held for a total period of time of about 30 hours to about 100 hours, or a total period of time of about 30 hours to about 90 hours, or a total period of time of about 30 hours to about 80 hours, or a total period of time of about 30 hours to about 70 hours, or a total period of time of about 30 hours to about 60 hours, or a total period of time of about 30 hours to about 50 hours.

In certain embodiments, drug substances comprising bispecific antibodies are gently mixed during thawing. Gentle mixing can be achieved, for example, by using a tilting shaker or by gently inverting the container of the drug substance. In certain embodiments, the drug substances comprising the bispecific antibody are not mixed during thawing, but rather the drug substances are gently mixed after thawing.

In certain embodiments, the drug substance comprises a bispecific antibody at a concentration of about 0.01mg/mL to about 25mg/mL, or about 0.05mg/mL to about 25mg/mL, about 0.1mg/mL to about 25mg/mL, about 0.5mg/mL to about 25mg/mL, or about 1mg/mL to about 25 mg/mL. In certain embodiments, the drug substance comprises a bispecific antibody at a concentration of about 1mg/mL to about 20mg/mL, or about 1mg/mL to about 15mg/mL, or about 1mg/mL to about 10mg/mL, or about 1mg/mL to about 5 mg/mL. In certain embodiments, the drug substance comprises a bispecific antibody at a concentration of about 0.01mg/mL, about 0.05mg/mL, about 0.1mg/mL, about 0.5mg/mL, about 1mg/mL, about 2mg/mL, about 3mg/mL, about 4mg/mL, about 5mg/mL, about 6mg/mL, about 7mg/mL, about 8mg/mL, about 9mg/mL, about 10mg/mL, about 11mg/mL, about 12mg/mL, about 13mg/mL, about 14mg/mL, about 15mg/mL, about 16mg/mL, about 17mg/mL, about 18mg/mL, about 19mg/mL, about 20mg/mL, or about 25 mg/mL.

In certain embodiments, the pH range of the drug substance comprising the bispecific antibody is from about pH 3.5 to about pH 7.5 or from about pH 4.0 to about pH 7.0. In certain embodiments, the pH of the drug substance comprising the bispecific antibody ranges from about pH 4.0 to about pH 6.5. In certain embodiments, the pH of the drug substance comprising the bispecific antibody ranges from about pH 4.0 to about pH 4.8. In certain embodiments, the pH of the drug substance comprising the bispecific antibody ranges from about 3.5, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6, about 6.2, about 6.4, about 6.6, about 7.0, or about 7.5.

In certain embodiments, the method further comprises filtering the drug substance. In certain embodiments, the filtering step comprises aseptic filtration. Sterile filtration is known and commonly used in the art. For example, sterile filtration may be performed using Normal Flow Filtration (NFF), in which the direction of fluid flow is perpendicular to the filtration media (e.g., membrane) and the purified liquid passes through the filtration media. In certain embodiments, agents that can reduce aggregation of the bispecific antibody (e.g., amino acids, polyols such as benzyl alcohol, cyclodextrins, dextran, and polyethylene glycol (PEG)) can be added to the drug substance.

In certain embodiments, the composition prepared by the method is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" is understood to refer to a formulation comprising a bispecific antibody suitable for injection and/or administration to a patient (e.g., a human) in need thereof. More particularly, the pharmaceutical composition is substantially sterile and free of any agent that is overly toxic or infectious to the recipient.

In certain embodiments, the method for making a composition comprising a bispecific antibody further comprises aliquoting the composition into a pharmaceutical product form. Such pharmaceutical product forms may be presented in unit dosage form (e.g., in ampoules, single dose containers, or multi-dose containers). These pharmaceutical product forms may, if desired, be presented in vials, pack or dispenser devices, which may contain one or more unit dosage forms containing the bispecific antibody.

In certain embodiments, the method for preparing a composition comprising a bispecific antibody further comprises lyophilizing the composition. In certain embodiments, the lyophilization step is performed after the composition is aliquoted into the form of a pharmaceutical product. Methods of lyophilizing pharmaceutical compositions are known and commonly used in the art. See, e.g., cryo-preservation and Freeze-Drying Protocols (edited by j.g.day and g.n.standard, Springer [ sipringer press ] 2017). The lyophilization step may be performed before or after the aliquoting step.

In certain embodiments, the method for making a composition comprising a bispecific antibody further comprises spray drying the composition. Methods of spray drying pharmaceutical compositions are known and commonly used in the art. For example, Niven, r., Prestrelski, s.j., Treuheit, m.j., Ip, a.y, and Arakawa, t.protein neutralization ii.stabilization of G-CSF to air-jet neutralization and the role of protectants, [ protein atomization ii.g-CSF stabilization of air atomization and the effect of protectants ] (1996) int.j.pharm. [ international journal of pharmaceuticals ]127,191-20. The spray drying step may be performed before or after the aliquoting step.

The methods disclosed herein reduce aggregation of bispecific antibodies. These methods are based on the surprising discovery that when thawed antibodies are held at a temperature (e.g., about 5 ℃ to about 45 ℃) for a period of at least 4 hours (e.g., a period of about 4 hours to about 96 hours), the aggregates formed during storage under freezing conditions (e.g., about-20 ℃ to about-40 ℃) are reduced. Without wishing to be bound by any theory, it is believed that the aggregates revert back to the unaggregated state after the retention period.

In certain embodiments, the aggregate comprises a HMW aggregate. In certain embodiments, the bispecific antibody comprises less than about 5%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.5% HMW aggregates after the retention period. In certain embodiments, HMW aggregates formed under freezing conditions are reduced to the same level or substantially the same level as before freezing. In certain embodiments, the HMW aggregate comprises a dimer of a bispecific antibody. In certain embodiments, the bispecific antibody comprises less than about 1% of dimers of the bispecific antibody after the retention period. In certain embodiments, the bispecific antibody comprises less than about 0.5% of dimers of the bispecific antibody after the retention period.

In certain embodiments, the bispecific antibody-containing drug substance comprises less than about 5%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.5% HMW aggregates after the retention period. In certain embodiments, the HMW aggregate comprises a dimer of a bispecific antibody. In certain embodiments, the drug substance comprising the bispecific antibody comprises less than about 1% of dimers of the bispecific antibody after the retention period. In certain embodiments, the drug substance comprising the bispecific antibody comprises less than about 0.5% dimers of the bispecific antibody after the retention period.

The methods disclosed herein have no or substantially no effect on the stability profile of the bispecific antibody or drug substance comprising the bispecific antibody. In certain embodiments, the methods produce a bispecific antibody or drug substance comprising a bispecific antibody that has the same or substantially the same color and/or transparency as the bispecific antibody or drug substance before freezing. In certain embodiments, the methods produce a bispecific antibody or drug substance comprising a bispecific antibody with the same or substantially the same charge variant as the bispecific antibody or drug substance before freezing. In certain embodiments, the methods produce a bispecific antibody or drug substance comprising a bispecific antibody with the same or substantially the same potency as the bispecific antibody or drug substance before freezing. In certain embodiments, the methods produce a bispecific antibody or drug substance comprising a bispecific antibody with the same or substantially the same level of cleavage as the bispecific antibody or drug substance prior to freezing. In certain embodiments, the methods produce a bispecific antibody or drug substance comprising a bispecific antibody with the same or substantially the same chemical modification (e.g., glycosylation) as the bispecific antibody or drug substance prior to freezing. In certain embodiments, the methods produce a bispecific antibody or drug substance comprising a bispecific antibody having the same or substantially the same pH as the bispecific antibody or drug substance prior to freezing.

Bispecific antibodies

Bispecific antibodies useful in the methods disclosed herein include those that tend to aggregate under freezing conditions, e.g., at temperatures in the range of-20 ℃ to-50 ℃ (e.g., due to hydrophobic interactions between different regions of the antibody). As used herein, the term "bispecific antibody" is understood to refer to an antibody capable of specifically binding two different antigens or targets or epitopes. In certain embodiments, the bispecific antibody comprises a first domain that specifically binds to one antigen or target and a second domain that specifically binds to another antigen or target. In certain embodiments, the first domain of the bispecific antibody specifically binds to a target cell surface antigen and the second binding domain of the bispecific antibody specifically binds to human CD3, a subunit of the T cell receptor complex on T cells. In certain preferred embodiments, the bispecific antibody is a bispecific T cell junction (BiTE) antibody construct. See, for example, WO 2008119567 and WO 2017134140.

As used herein below, the term "domain-specific binding" or "domain binding" is understood to refer to a domain that specifically binds/interacts/recognizes a given target or epitope. The binding domain of the antibody construct comprises the minimal structural requirements of the antibody that allow target binding. Such minimum requirements may be defined by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the light chain variable region (VL)) and/or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the heavy chain variable region (VH)), preferably all six CDRs. Preferably, those CDRs are contained in the framework of the antibody VL and antibody VH. The term bagFragments of full-length antibodies and antibody variants are included. Examples of antibody fragments, antibody variants, or binding domains include (1) Fab fragments, a monovalent fragment having VL, VH, CL, and CH1 domains; (2) f (ab')₂A fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) (ii) an Fv fragment having VL and VH domains of a single arm of an antibody; (5) dAb fragments with VH Domain (Ward et al, (1989) Nature [ Nature]341: 544-; (6) isolated Complementarity Determining Regions (CDRs), and (7) single chain fv (scFV), the latter being preferred (e.g., derived from a scFV library). Additional antibody fragments include VH, VHH, VL,(s) dAb, Fab', and "r IgG" ("half antibodies").

The term "antibody construct" is understood to mean a molecule wherein the structure and/or function is based on the structure and/or function of an antibody, e.g. a full-length or whole immunoglobulin molecule, and/or is extracted from the variable heavy chain (VH) and/or variable light chain (VL) domains of an antibody or fragment thereof. Thus, the antibody construct is capable of binding to its specific target or antigen. The antibody constructs also include modified antibody fragments (also referred to as antibody variants, such as scFv, bis-scFv or bis(s) -scFv, scFv-Fc, scFv-zippers, scFab, Fab₂、Fab₃Diabodies, single chain diabodies, tandem diabodies (Tandab), tandem diabodies, tandem triabodies, "such as triabodies or tetrabodies, and single domain antibodies (such as nanobodies or single variable domain antibodies, which may specifically bind an antigen or epitope independently of other V regions or domains) comprising only one variable domain (which may be VHH, VH or VL).

As used herein, the term "single chain Fv", "single chain antibody" or "scFv" is understood to refer to a single polypeptide chain antibody fragment that comprises variable regions from both the heavy and light chains but lacks constant regions. Typically, the single chain antibody further comprises a polypeptide linker between the VH and VL domains which enables the single chain antibody to form the desired structure which allows antigen binding. Single chain Antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies [ Pharmacology of Monoclonal Antibodies ], Vol.113, Rosenburg and Moore, eds Springer-Verlag [ Schpringer Press ], New York, p.269-315 (1994). Various methods of producing single chain antibodies are known, including those described in: U.S. Pat. nos. 4,694,778 and 5,260,203; international patent application publication Nos. WO 88/01649; bird (1988) Science [ Science ]242: 423-; huston et al (1988) Proc. Natl.Acad.Sci.USA [ Proc. Natl.Acad.Sci ]85: 5879-; ward et al (1989) Nature [ Nature ]334: 54454; skerra et al (1988) Science 242: 1038-1041. In particular embodiments, single chain antibodies may also be bispecific human and/or humanized and/or synthetic.

In certain embodiments, the first and second domains of the bispecific antibody are "bispecific single chain antibody constructs", more preferably bispecific "single chain Fv" (scFv). Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker (as described herein) that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules; see, for example, Huston et al (1988) Proc. Natl. Acad. Sci USA [ Proc. Natl.Acad. Sci ]85: 5879-. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the function of the fragments is assessed in the same manner as for intact or full-length antibodies. Thus, an scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, typically linked to a short linker peptide of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is typically glycine rich for flexibility and serine or threonine for solubility, and may link the N-terminus of the VH with the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the scFv retains the specificity of the original immunoglobulin.

Bispecific single chain variable fragment (with (scFv)₂Format di-scFv or di-scFv) can be engineered by linking two scFv molecules (e.g., using a linker as described above). Ligation can be performed by generating a single peptide chain with two VH regions and two VL regions, thereby generating tandem scFv (see, e.g., Kufer P. et al, (2004) Trends in Biotec)Technology (Biotechnology trends)]22(5):238-244). Another possibility is to generate scFv molecules with linker peptides that are too short for the two variable regions to fold together (e.g., about five amino acids), forcing scFv dimerization. This type is known as diabodies (see, e.g., Hollinger, Philipp et al, (7. 1993) Proceedings of the National Academy of Sciences of the United States of America [ Proc. Natl. Acad. Sci. USA ] A.]90(14):6444-8)。

In certain embodiments, the first domain and the second domain of the bispecific antibody specifically bind to the target cell surface antigen and human CD3, respectively. In certain embodiments, the first domain and the second domain of the bispecific antibody form a bispecific antibody construct in a form selected from the group consisting of: (scFv)₂scFv-single domain mAbs, diabodies and oligomers of any of those formats.

In certain embodiments, the first, second, or first and second domains may comprise a single domain antibody, which is the variable domain or at least the CDR, respectively, of a single domain antibody. Single domain antibodies comprise only one (monomeric) antibody variable domain that is capable of selectively binding to a particular antigen independently of other V regions or domains. The first single domain antibodies are engineered from heavy chain antibodies found in camelids, and these are referred to as VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR) from which a heavy chain called V can be obtained_NARA single domain antibody of a fragment. An alternative approach is to split the dimeric variable domain from a common immunoglobulin (e.g. from a human or rodent) into monomers, thereby obtaining VH or VL as single domain Ab. While most studies on single domain antibodies are currently based on heavy chain variable domains, nanobodies derived from light chains also show specific binding to a target epitope. Examples of single domain antibodies are so-called sdabs, nanobodies or single variable domain antibodies.

In certain embodiments, the first (binding) domain of the bispecific antibody binds to a target cell surface antigen. In some embodiments, the target cell surface antigen is CD 70. CD70 (also known as CD27L or TNFSF7) is a type II integral membrane protein whose normal expression is restricted to a subset of activated T and B cells, mature dendritic cells and thymic medullary epithelial cells.

In other embodiments, the target cell surface antigen is a tumor antigen. As used herein, the term "tumor antigen" is understood to refer to those antigens that are presented on tumor cells. These antigens may be presented on the cell surface as extracellular portions, which are typically combined with the transmembrane and cytoplasmic portions of the molecule. These antigens are sometimes only presented by tumor cells and not by normal cells. Tumor antigens may be expressed only on tumor cells or may represent tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. More common are antigens presented by tumor cells and normal cells, and they are referred to as tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells or, since the structure of tumor tissue is less compact compared to normal tissue, facilitate antibody binding in tumor cells. In some embodiments, the second (binding) domain binds to a tumor antigen selected from the group consisting of: CD19, CD33, epidermal growth factor receptor variant iii (egfrviii), Mesothelin (MSLN), cadherin 19(CDH19), FMS-like tyrosine kinase 3(FLT3), delta-like ligand 3(DLL3), placental cadherin (CDH3), B Cell Maturation Antigen (BCMA), or Prostate Specific Membrane Antigen (PSMA). In some embodiments, the tumor antigen is a human tumor antigen.

In certain embodiments, the second (binding) domain of the bispecific antibody binds to human CD3 epsilon on the surface of a T cell. In certain preferred embodiments, the second domain of the bispecific antibody binds to an extracellular epitope of the epsilon chain of human CD 3. In some preferred embodiments, the second domain of the bispecific antibody that binds to an extracellular epitope of human CD3 comprises a VL region comprising a CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of:

(a) CDR-L1 as depicted in SEQ ID NO:27 of WO 2008/119567, CDR-L2 as depicted in SEQ ID NO:28 of WO 2008/119567 and CDR-L3 as depicted in SEQ ID NO:29 of WO 2008/119567;

(b) CDR-L1 as depicted in SEQ ID NO:117 of WO 2008/119567, CDR-L2 as depicted in SEQ ID NO:118 of WO 2008/119567 and CDR-L3 as depicted in SEQ ID NO:119 of WO 2008/119567; and

(c) CDR-L1 as depicted in SEQ ID NO:153 of WO 2008/119567, CDR-L2 as depicted in SEQ ID NO:154 of WO 2008/119567 and CDR-L3 as depicted in SEQ ID NO:155 of WO 2008/119567.

In another preferred embodiment, the second domain of the bispecific antibody binds to an extracellular epitope of the epsilon chain of human CD3 and comprises a VH region comprising a CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of:

(a) CDR-H1 as depicted in SEQ ID NO 12 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO 13 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO 14 of WO 2008/119567;

(b) CDR-H1 as depicted in SEQ ID NO:30 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:31 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:32 of WO 2008/119567;

(c) CDR-H1 as depicted in SEQ ID NO:48 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:49 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:50 of WO 2008/119567;

(d) CDR-H1 as depicted in SEQ ID NO:66 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:67 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:68 of WO 2008/119567;

(e) CDR-H1 as depicted in SEQ ID NO:84 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:85 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:86 of WO 2008/119567;

(f) CDR-H1 as depicted in SEQ ID NO:102 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:103 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:104 of WO 2008/119567;

(g) CDR-H1 as depicted in SEQ ID NO:120 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:121 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:122 of WO 2008/119567;

(h) CDR-H1 as depicted in SEQ ID NO:138 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:139 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:140 of WO 2008/119567;

(i) CDR-H1 as depicted in SEQ ID NO:156 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:157 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:158 of WO 2008/119567; and

(j) CDR-H1 as depicted in SEQ ID NO:174 of WO 2008/119567, CDR-H2 as depicted in SEQ ID NO:175 of WO 2008/119567 and CDR-H3 as depicted in SEQ ID NO:176 of WO 2008/119567.

In certain preferred embodiments, the aforementioned three sets of VL CDRs are combined with the aforementioned ten sets of VH CDRs within the second binding domain to form (30) sets, each set comprising CDRs 1-3.

It is also preferred that the second domain which binds to CD3 comprises a VH region selected from the group of VH regions consisting of: as depicted in SEQ ID NO 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008/119567 or as depicted in SEQ ID NO 15 or 24 of the present sequence Listing.

Preferably, the second domain that binds to CD3 comprises a VL region selected from the group of VL regions consisting of: such as depicted in SEQ ID NO 17, 21, 35, 39, 53, 57, 71, 75, 89, 93, 107, 111, 125, 129, 143, 147, 161, 165, 179 or 183 of WO 2008/119567 or as depicted in SEQ ID NO 16 or 25 of the present sequence Listing.

More preferably, the bispecific antibody is characterized by a second domain that binds CD3, the second domain comprising a VL region and a VH region selected from the group consisting of:

(a) a VL region as depicted in SEQ ID NO 17 or 21 of WO 2008/119567 and a VH region as depicted in SEQ ID NO 15 or 19 of WO 2008/119567;

(b) a VL region as depicted in SEQ ID NO 35 or 39 of WO 2008/119567 and a VH region as depicted in SEQ ID NO 33 or 37 of WO 2008/119567;

(c) a VL region as depicted in SEQ ID NO 53 or 57 of WO 2008/119567 and a VH region as depicted in SEQ ID NO 51 or 55 of WO 2008/119567;

(d) a VL region as depicted in SEQ ID NO:71 or 75 of WO 2008/119567 and a VH region as depicted in SEQ ID NO:69 or 73 of WO 2008/119567;

(e) a VL region as depicted in SEQ ID NO:89 or 93 of WO 2008/119567 and a VH region as depicted in SEQ ID NO:87 or 91 of WO 2008/119567;

(f) a VL region as depicted in SEQ ID NO:107 or 111 of WO 2008/119567 and a VH region as depicted in SEQ ID NO:105 or 109 of WO 2008/119567;

(g) a VL region as depicted in SEQ ID NO 125 or 129 of WO 2008/119567 and a VH region as depicted in SEQ ID NO 123 or 127 of WO 2008/119567;

(h) a VL region as depicted in SEQ ID NO:143 or 147 of WO 2008/119567 and a VH region as depicted in SEQ ID NO:141 or 145 of WO 2008/119567;

(i) a VL region as depicted in SEQ ID NO:161 or 165 of WO 2008/119567 and a VH region as depicted in SEQ ID NO:159 or 163 of WO 2008/119567; or

(j) A VL region as depicted in SEQ ID NO:179 or 183 of WO 2008/119567 and a VH region as depicted in SEQ ID NO:177 or 181 of WO 2008/119567.

Also in preferred embodiments, the bispecific antibody comprises a second domain that binds to CD3, the second domain comprising a VL region as depicted in SEQ ID NO 16 or 25 and a VH region as depicted in SEQ ID NO 15 or 24 of the present sequence listing.

A preferred embodiment of the bispecific antibody described above is characterized in that the second domain that binds CD3 comprises an amino acid sequence selected from the group consisting of: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 of WO 2008/119567 or as depicted in SEQ ID NO 26 in this sequence Listing.

According to a preferred embodiment, the firstOne domain and/or the second domain has the following form: the pair of VH and VL regions is in the form of a single chain antibody (scFv). The VH and VL regions are arranged in the order VH-VL or VL-VH. Preferably, the VH domain is located at the N-terminus and the VL domain is located at the C-terminus of the linker sequence. In certain embodiments, the first domain and the second domain of the bispecific antibody form a bispecific antibody in a form selected from: (scFv)₂scFv-single domain mAb, diabody or oligomer of any of those formats.

In certain preferred embodiments, the bispecific antibody further comprises a third domain. In certain embodiments, the third domain is a single chain fc (scfc) domain. In certain preferred embodiments, the scFc domain is a scFc half-life extended (HLE) domain.

The term "Fc" moiety or "Fc" monomer is understood to refer to a polypeptide comprising at least one domain having the function of the CH2 domain and at least one domain having the function of the CH3 domain of an immunoglobulin molecule. Polypeptides comprising those CH domains are "polypeptide monomers". The Fc monomer may be a polypeptide comprising at least a fragment of a constant region of an immunoglobulin, excluding the first constant region immunoglobulin domain of a heavy chain (CH1), but retaining at least a functional portion of a CH2 domain and a functional portion of a CH3 domain, wherein the CH2 domain is amino terminal to the CH3 domain. In a preferred embodiment, the Fc monomer may be a polypeptide constant region comprising a portion of an Ig-Fc hinge region, a CH2 region, and a CH3 region, wherein the hinge region is the amino-terminus of the CH2 domain. It is believed that the hinge region of the bispecific antibody promotes dimerization. Such Fc polypeptide molecules can be obtained, for example, by papain digestion of immunoglobulin regions (which, of course, produce a dimer of two Fc polypeptides). In another embodiment, the Fc monomer may be a polypeptide region comprising a CH2 region and a portion of a CH3 region. Such Fc polypeptide molecules may be obtained by, for example, pepsin digestion of immunoglobulin molecules. In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequence of: an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgM Fc region, an IgA Fc region, an IgD Fc region, and an IgE Fc region. (see, e.g., Padlan, Molecular Immunology, 31(3), 169. sup. 217 (1993)). In one embodiment, the Fc monomer has an amino acid sequence as disclosed in WO 2014/153063. As there are some variations between immunoglobulins, and for clarity only, Fc monomers are understood to refer to the last two heavy chain constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM. As noted above, the Fc monomer may also include the flexible hinge N-terminus of these domains. For IgA and IgM, the Fc monomers may include J chains. For IgG, the Fc portion comprises the immunoglobulin domains CH2 and CH3 and the hinge between the first two domains and CH 2. Although the boundaries of the Fc portion may vary, an example of a human IgG heavy chain Fc portion comprising a functional hinge, CH2 and CH3 domains may be defined as, for example, comprising residues D231 (residues of the hinge domain-corresponding to D234 in table 1 below) to the carboxy-terminal P476 of the CH3 domain, respectively L476 (for IgG4), where the numbering is according to Kabat. The two Fc moieties or Fc monomers fused to each other via a peptide linker define a third domain of the antibody construct of the invention, which may also be defined as an scFc domain.

The IgG hinge region can be identified by analogy using the Kabat numbering listed in table 1. It is envisaged that the hinge domain/region of the third domain comprises amino acid residues corresponding to the IgG1 sequence stretch of D234 to P243 according to Kabat numbering. It is also envisaged that the hinge domain/region of the third domain comprises or consists of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO:191) (variations of said sequence are also envisaged corresponding to the segments D234 to P243 shown in table 1 below, as long as the hinge region still promotes dimerization). In a preferred embodiment, the glycosylation site in the third domain of the antibody construct at Kabat position 314 of the CH2 domain is removed by a N314X substitution, wherein X is any amino acid except Q. The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intra-domain cysteine disulfide bridges at Kabat positions 309 and 321).

Table 1: kabat numbering of amino acid residues in the hinge region

In some embodiments, the hinge domain/region comprises or consists of: an IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO:192), an IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO:193) or ELKTPLGDTTHTCPRCP (SEQ ID NO:194) and/or an IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 195). The IgG1 subtype hinge sequence may be one of the following EPKSCDKTHTCPPCP (shown in Table 1 and SEQ ID NO: 196). Thus, these core hinge regions are also contemplated in the context of bispecific antibodies.

The positions and sequences of the IgG CH2 and IgG CD3 domains can be identified by analogy using the Kabat numbering listed in table 2:

table 2: kabat numbering of amino acid residues in IgG CH2 and CH3 regions

In one embodiment, the bolded amino acid residues highlighted in the CH3 domain of the first or both Fc monomers are deleted.

If a linker is used to fuse the first domain to the second domain or to fuse the first domain or the second domain to the third domain, the linker is preferably of a length and sequence sufficient to ensure that each of the first and second domains is able to retain their differential binding specificities independently of each other. For peptide linkers connecting at least two binding domains (or two variable domains) in a bispecific antibody construct, peptide linkers comprising only a small number of amino acid residues, e.g. 12 amino acid residues or less, are preferred. Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. Contemplated peptide linkers having less than 5 amino acids comprise 4, 3, 2 or 1 amino acids, with Gly-rich linkers being preferred.

A particularly preferred "single" amino acid "peptide linker" is Gly. Thus, a peptide linker may consist of a single amino acid Gly. In a preferred embodiment, the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser (i.e., Gly4Ser (SEQ ID NO:197)) or a polymer thereof (i.e., (Gly4Ser) x, wherein x is an integer of 1 or more (e.g., 2 or 3)). In another preferred embodiment, the peptide linker has the amino acid sequence Gly-Gly-Gly-Gly-Ser (i.e., Gly4Ser (SEQ ID NO:197)) or a polymer thereof (i.e., (Gly4Ser) x, wherein x is an integer of 5 or more (e.g., 5, 6, 7, 8, etc., or more). In certain embodiments, x is preferably 6((Gly4Ser) 6). The characteristics of peptide linkers (including the absence of facilitation of secondary structure) are known in the art and described, for example, in the following documents: dall' Acqua et al (Biochem. [ biochemistry ] (1998)37, 9266-. Peptide linkers that do not promote any secondary structure are preferred. Methods for preparing fused and operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99/54440 or Sambrook et al, Molecular Cloning: A Laboratory Manual [ Molecular Cloning: A Laboratory Manual ], Cold Spring Harbor Laboratory Press [ Cold Spring Harbor Laboratory Press ], Cold Spring Harbor, New York [ Cold Spring Harbor, N.Y. ], 2001).

A preferred embodiment of a peptide linker for fusion of the first domain and the second domain has the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO: 197). Preferred examples of linkers for peptide linkers for the fusion of the second and third domains are (Gly) 4-linkers, respectively G4-linkers.

The peptide linker at which the polypeptide monomers of the third domain ("Fc portion" or "Fc monomer") are fused to each other preferably comprises at least 25 amino acid residues (25, 26, 27, 28, 29, 30, etc.). More preferably, the peptide linker comprises at least 30 amino acid residues (30, 31, 32, 33, 34, 35, etc.). The linker also preferably comprises up to 40 amino acid residues, more preferably up to 35 amino acid residues, most preferably exactly 30 amino acid residues. Preferred embodiments of such peptide linkers are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser (i.e., Gly4Ser (SEQ ID NO:197)) or a polymer thereof (i.e., (Gly4Ser) x, wherein x is an integer of 5 or more (e.g., 6, 7 or 8)). The integer is preferably 6 or 7, and the integer is more preferably 6.

In some preferred embodiments, the third domain of the bispecific antibody is an HLE domain having an amino to carboxy order:

hinge-CH 2-CH 3-linker-hinge-CH 2-CH 3.

In certain embodiments, one of the third domains, or preferably the CH2 domain of each (two) polypeptide monomer, comprises a cysteine disulfide bridge within the domain. The term "cysteine disulfide bridge" refers to a functional group having the general structure R-S-S-R, as is known in the art. This linkage is also known as an SS bond or a disulfide bridge and is derivatized by coupling two thiol groups of cysteine residues. For bispecific antibodies, it is particularly preferred to introduce cysteine, which forms a cysteine disulfide bridge in the mature antibody construct, into the amino acid sequence corresponding to the CH2 domains of 309 and 321(Kabat numbering).

In one embodiment, the glycosylation site in Kabat position 314 of the CH2 domain is removed. Removal of the glycosylation site is preferably achieved by a N314X substitution, wherein X is any amino acid except Q. The substitution is preferably an N314G substitution. In a more preferred embodiment, the CH2 domain further comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intra-domain cysteine disulfide bridges at Kabat positions 309 and 321).

It is assumed that the preferred features of bispecific properties compared to e.g. bispecific heterogeneous Fc antibody constructs known in the art may especially involve the introduction of the above mentioned modifications in the CH2 domain. Thus, it is preferred that the CH2 domain in the third domain of the bispecific antibody comprises a cysteine disulfide bridge within the domain at Kabat positions 309 and 321 and/or a glycosylation site at Kabat position 314 is removed by an N314X substitution, preferably by an N314G substitution, as described above.

In a further preferred embodiment, the CH2 domain in the third domain of the bispecific antibody comprises intra-domain cysteine disulfide bridges at Kabat positions 309 and 321, and the glycosylation site at Kabat position 314 is removed by an N314G substitution.

In certain embodiments, the third domain of the bispecific antibody comprises or consists of, in amino to carboxy order: DKKHTTCPPCP (SEQ ID NO:191) (i.e., hinge) -CH2-CH 3-linker-DKKHTTCPPCP (SEQ ID NO:191) (i.e., hinge) -CH2-CH 3. In a preferred embodiment, the peptide linker of the above-described bispecific antibody is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser (SEQ ID NO:197), or a multimer thereof, i.e., (Gly4Ser) x, wherein x is an integer of 5 or more (e.g., 5, 6, 7, 8, etc., or more), preferably 6((Gly4Ser) 6). The antibody may further comprise the above-described substitutions N314X, preferably N314G, and/or other substitutions V321C and R309C.

Bispecific antibodies may also comprise additional domains that, for example, aid in the isolation of the molecule or relate to adaptive pharmacokinetic characteristics of the molecule. The domains that facilitate isolation of the antibody construct may be selected from peptide motifs or adjuvantly introduced moieties that can be captured in a separation method, such as a separation column. Non-limiting examples of such additional domains include peptide motifs known as Myc-tags, HAT-tags, HA-tags, TAP-tags, GST-tags, chitin binding domains (CBD-tags), maltose binding proteins (MBP-tags), Flag-tags, Strep-tags and variants thereof (e.g., strepII-tags) and His-tags. All bispecific antibodies disclosed herein may comprise a His-tag domain commonly referred to as a contiguous His-residue repeat of preferably 5, and more preferably 6 His residues (hexa-histidine) in the amino acid sequence of the molecule. The His tag may be located, for example, at the N-or C-terminus of the antibody construct, which is preferably located at the C-terminus. Most preferably, the hexahistidine tag (HHHHHHHHHH) (SEQ ID NO:198) is linked to the C-terminus of the bispecific antibody via a peptide bond. In addition, the conjugate system of PLGA-PEG-PLGA may be combined with a polyhistidine tag for sustained release applications and improved pharmacokinetic profiles.

In certain embodiments, the bispecific antibody comprises a first domain and a second domain, wherein:

(i) the first domain comprises two antibody variable domains and the second domain comprises two antibody variable domains;

(ii) the first domain comprises one antibody variable domain and the second domain comprises two antibody variable domains;

(iii) the first domain comprises two antibody variable domains and the second domain comprises one antibody variable domain; or

(iv) The first domain comprises an antibody variable domain and the second domain comprises an antibody variable domain.

Thus, the first and second domains may be binding domains each comprising two antibody variable domains, such as a VH and VL domain. Examples of such binding domains comprising two antibody variable domains are described above and comprise, for example, an Fv fragment, an scFv fragment or an Fab fragment as described above. Alternatively, one or both of these binding domains may comprise only a single variable domain. Examples of such single domain binding domains are described above and include, for example, nanobodies or single variable domain antibodies comprising only one variable domain (which may be VHH, VH or VL) that specifically binds an antigen or epitope independently of the other V regions or domains.

In some preferred embodiments, the bispecific antibody comprises a first domain, a second domain, and a third domain, wherein the first domain binds to CD70, the second domain binds to human CD3, and the third domain is an HLE domain having an amino to carboxyl order: hinge-CH 2-CH 3-linker-hinge-CH 2-CH 3. In other preferred embodiments, the bispecific antibody comprises a first domain, a second domain and a third domain, wherein the first domain binds to a tumor antigen selected from the group consisting of: CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, BCMA, or PSMA, the second domain binding to human CD3, and the third domain being an HLE domain having an amino to carboxyl order: hinge-CH 2-CH 3-linker-hinge-CH 2-CH 3. In a preferred embodiment, the first domain and the second domain are fused to the third domain by a peptide linker. Preferred peptide linkers have been described above and are characterized by the amino acid sequence Gly-Ser, i.e. Gly4Ser, or a polymer thereof, i.e. (Gly4Ser) x, wherein x is an integer of 1 or more (e.g. 2, 3, 4, 5, 6 or 7).

In some embodiments, the bispecific antibody is characterized by having an amino acid sequence selected from the group consisting of:

(a) 37 to 41 of SEQ ID NO; CD33

(b) 51 and 52; EGFRvIII

(c) 62, 63 and 64; MSLN

(d) 74 to 82 CDH19 of SEQ ID NO

(e) 103 and 104 DLL3 SEQ ID NOs

(f) 17, 113 and 114 CD19 SEQ ID NOs

(g) 92 and 93 FLT3 SEQ ID NO

(h) 124 and 125 CDH3 SEQ ID NO

(i) 135 and 136 BCMA SEQ ID NO

(j) 146 to 151, 161 to 168 and 176 to 181PSMA or SEQ ID NO

(k) 188 to 190 CD70 SEQ ID NO

Any of the foregoing bispecific antibodies may or may not have a third domain that is an extended half-life (HLE) domain that is preferably a scFc domain or a heterogeneous Fc domain or an albumin binding domain. The second domain of a bispecific antibody that binds human CD3 can be linked to the N-terminus or C-terminus of the HLE domain (e.g., via a linker as described above).

In some embodiments, the bispecific antibody is a CD70xCD3 bispecific antibody comprising a first domain that binds to CD70 and a second domain that binds to CD 3. In one embodiment, the first domain binds to CD70 and has the CDRs depicted in SEQ ID NOs 182 to 187, and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the bispecific antibody comprises, consists essentially of, or consists of a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:188 and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 189. In one embodiment, the CD70xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 190.

In some embodiments, the bispecific antibody is a CD19xCD3 bispecific antibody comprising a first domain that binds to CD19 and a second domain that binds to CD 3. In one embodiment, the first domain binds to CD19 and has the CDRs depicted in SEQ ID NOs 1 to 6 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In another embodiment, the first domain binds to CD19 and has the CDRs depicted in SEQ ID NOs 105 to 107 and 109 to 111, the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14, and further comprises an HLE domain (third domain). In one embodiment, the CD19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 17. In another embodiment, the CD19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 114.

In some embodiments, the bispecific antibody is a BCMAxCD3 bispecific antibody comprising a first domain that binds BCMA and a second domain that binds CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to BCMA and has the CDRs as depicted in SEQ ID NOs 126 to 131 and the second domain binds to CD3 and has the CDRs as depicted in SEQ ID NOs 9 to 14. In one embodiment, the BCMAxCD3 bispecific antibody comprises, consists essentially of, or consists of a VH comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 132 and a VL comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 133. In one embodiment, the BCMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 135. In another embodiment, the BCMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 136.

In some embodiments, the bispecific antibody is a CD33xCD3 bispecific antibody comprising a first domain that binds to CD33 and a second domain that binds to CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to CD33 and has the CDRs depicted in SEQ ID NOs 29 to 31 and 34 to 36 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the CD33xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO 27 or 28, and the VL comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO 32 or 33. In one embodiment, the CD33xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 40. In another embodiment, the BCMAXCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 41.

In some embodiments, the bispecific antibody is an EGFRvIII xcd3 bispecific antibody comprising a first domain that binds to EGFRvIII and a second domain that binds to CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to EGFRvIII and has the CDRs depicted in SEQ ID NOs 42 to 47 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the egfrviii xcd3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 48 and a VL comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 49. In one embodiment, the egfrviii xcd3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 52.

In some embodiments, the bispecific antibody is a MSLNxCD3 bispecific antibody comprising a first domain that binds to MSLN and a second domain that binds to CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to MSLN and has the CDRs depicted in SEQ ID NOs 53 to 58 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the MSLNxCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO:59 and the VL comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 60. In one embodiment, the MSLNxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 63. In one embodiment, the MSLNxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 64.

In some embodiments, the bispecific antibody is a CDH19xCD3 bispecific antibody comprising a first domain that binds to CDH19 and a second domain that binds to CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to CDH19 and has the CDRs depicted in SEQ ID NOs 65 to 70 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the CDH19xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 71 and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO 72. In one embodiment, the CDH19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs 74-82. In one embodiment, the CDH19xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 82.

In some embodiments, the bispecific antibody is a DLL3xCD3 bispecific antibody comprising a first domain that binds to DLL3 and a second domain that binds to CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to DLL3 and has the CDRs depicted in SEQ ID NOs 94 to 99 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the DLL3xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 100, and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 101. In one embodiment, the DLL3xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 104.

In some embodiments, the bispecific antibody is a FLT3xCD3 bispecific antibody comprising a first domain that binds FLT3 and a second domain that binds CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to FLT3 and has the CDRs depicted in SEQ ID NOs 83 to 88 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the FLT3xCD3 bispecific antibody comprises, consists essentially of, or consists of a VH comprising, the amino acid sequence of SEQ ID No. 89 and a VL comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 90. In one embodiment, the FTL3xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the bispecific antibody is a CDH3xCD3 bispecific antibody comprising a first domain that binds to CDH3 and a second domain that binds to CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to CDH3 and has the CDRs depicted in SEQ ID NOs 115 to 120 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the CDH3xCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 121 and the VL comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID No. 122. In one embodiment, the CDH3xCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 125.

In some embodiments, the bispecific antibody is a PSMAxCD3 bispecific antibody comprising a first domain that binds PSMA and a second domain that binds CD 3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain binds to PSMA and has the CDRs depicted in SEQ ID NOs 137 to 142 and the second domain binds to CD3 and has the CDRs depicted in SEQ ID NOs 9 to 14. In one embodiment, the PSMAxCD3 bispecific antibody comprises, consists essentially of, or consists of a VH comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 143 and a VL comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO 144. In one embodiment, the PSMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs 146 to 151, 161 to 168, and 176 to 181. In one embodiment, the PSMAxCD3 bispecific antibody comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 177.

Bispecific antibodies disclosed herein can be prepared by methods known in the art. For example, bispecific antibodies can be prepared by the methods disclosed in WO 2008/119657 and WO 2017/134140.

In some embodiments, the bispecific antibody is a bispecific masked antigen binding protein. Bispecific masked antigen binding proteins have been described previously. See, for example, international publication nos. WO 2017/040344; U.S. patent publication No. 2015/0079088. "bispecific masked antigen binding protein" or "masked bispecific binding protein" is understood to mean a masked antigen binding protein that binds two different antigens or epitopes. A "masked antigen binding protein" is a protein that includes a Masking Domain (MD) coupled (e.g., by a covalent bond or linker) to an antigen binding domain (AB) such that coupling of the MD inhibits or reduces binding of the AB to its antigen. The MD further comprises a protein recognition site (PR) comprising a substrate or binding site for a protein or protease such that the AB domain binds to an antigen or increases or induces binding of the AB domain to an antigen when the protein or protease binds to and/or cleaves the protein recognition site. Masked antigen binding proteins have been previously described in, for example, international publication No. WO 2017/040344, U.S. patent No. 9540440, U.S. publication No. 20150118254, U.S. patent No. 9127053, U.S. patent No. 9517276, and U.S. patent No. 8563269. It will be apparent to the skilled artisan that, in some embodiments, the masked antigen binding protein may lack MD due to cleavage of the PR by a protease, which results in release of at least MD (e.g., where MD is not linked to the masked antigen binding protein by a covalent bond (e.g., a disulfide bond between cysteine residues)).

In some embodiments, the masked antigen binding protein is Probody (as described, for example, in Polu KR and Lowman HB. expert Opin Biol Ther. [ expert opinion for biological therapy ]8 months 2014; 14(8): 1049-53; or Desnoyers LR et al, Sci Transl Med. [ scientific transformation medicine ]2013, 10 months 16 days; 5(207)) or ProTIA prodrugs (as described, for example, in Schellenber V.Amunix unverils next-generation immuno-pharmacological cancer therapy platform. [ Amunix corporation published next generation immuno-immunological tumor cancer therapy platform ] htps:// biopharmanales tablets Nature. 2016. 9 months, B20; see also http:// www.amunix.com/technology-9/. A). In some embodiments, the masked antigen binding protein has the following structural arrangement from N-terminus to C-terminus: MD-AB or AB-MD. In some embodiments, the masked antigen binding protein comprises a Linker Peptide (LP), and the masked antigen binding protein has the following structural arrangement from N-terminus to C-terminus: MD-LP-AB or AB-LP-MD.

When the masked antigen-binding protein is in the presence of an antigen in its native state, binding of the AB to the antigen is reduced or inhibited as compared to binding of the AB to the antigen when the masked antigen-binding protein is in an active state (i.e., when a protein or protease binds to the PR and/or removes or translocates the MD). The ability of a masked antigen-binding protein to bind an antigen in its native state is reduced, e.g., by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even about 100%, when compared to the binding of an AB that is not associated with an MD (i.e., when the masked antigen-binding protein is in an active state), when measured in an in vitro and/or in vivo binding assay.

In some embodiments, the protein recognition site (PR) functions as a substrate for a protease, preferably an extracellular protease. PR can be selected based on proteins or proteases produced by cells proximal to the cells expressing the antigen (including, e.g., tumor cells) and/or by cells co-localized in the tissue (including, e.g., tumor cells) with the desired antigen of the AB of the masked antigen binding protein. In some embodiments, the protease is a u-type plasminogen activator (uPA, also known as urokinase), legumain, and/or a proteolytic enzyme (also known as MT-SP1 or MTSP 1). In some embodiments, the protease is a Matrix Metalloproteinase (MMP). In some embodiments, the protease is one of the proteases described in Rawlings, N and Salvesen, G.handbook of Proteolytic Enzymes (third edition). Esseville, Inc. (Elsevier),2013.ISBN: 978-0-12-382219-2.

In some embodiments, one antibody or antigen-binding fragment thereof (AB1) domain of the bispecific antigen-binding protein is specific for a target antigen and the other antibody or antigen-binding fragment thereof (AB2) domain is specific for another target antigen. In some embodiments, one antibody or antigen-binding fragment thereof (AB1) domain of the bispecific antigen-binding protein is specific for an epitope of a target antigen and the other antibody or antigen-binding fragment thereof (AB2) domain is specific for another epitope of the same target antigen.

The term "antibody fragment," "antibody fragment thereof," or "antigen-binding antibody fragment" is understood to refer to a portion of an intact antibody. "antigen-binding fragment" or "antigen-binding fragment thereof" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the epitope variable region of an intact antibody. Examples of antibody fragment antigen binding fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments, linear antibodies, scFv, and single chain antibodies.

The bispecific masked antigen binding protein comprises at least one Masking Domain (MD) comprising a protein recognition site (PR), wherein the MD inhibits or reduces the binding of AB to the antigen. The at least one MD comprises a protein recognition site (PR) comprising a substrate or binding site for a protein or protease such that when the protein or protease binds and/or cleaves the protease recognition site, the AB domain binds antigen or binding is increased. For bispecific masked antigen binding proteins, the masked antigen binding protein preferably comprises two MDs (e.g., MD1 and MD2) that reduce the ability of each antigen binding domain (AB1 and AB2) to bind its respective antigen or epitope.

The masked bispecific masked antigen binding proteins provided herein are stable in circulation and are activated at the intended site of therapy and/or diagnosis, but not in normal (i.e., healthy) tissue. When in the active state, the masked antigen binding protein and/or the bispecific or multispecific masked antigen binding protein exhibits binding to an antigen that is at least comparable to the binding of the corresponding unmodified antibody or bispecific or multispecific antigen binding protein (i.e., the corresponding antibody (counter) that does not comprise a masking domain) to an antigen.

In various embodiments, the bispecific masked antigen binding proteins described herein bind to human CD 3. For example, in various aspects, bispecific masked antigen binding proteins activate T cells by engagement of CD3 epsilon on T cells. That is, the antibody agonizes, stimulates, activates and/or enhances CD 3-mediated T cell activation. Biological activities of CD3 include, for example, T cell activation and other signaling through the interaction between CD3 and the antigen binding subunit of the T Cell Receptor (TCR).

Bispecific masked antigen binding proteins disclosed herein optionally bind to CD3 epsilon with a binding constant (Kd) of ≦ 1 μ M, e.g., in some embodiments, 100nM ≦ 10nM, or ≦ 1 nM.

In various aspects, the bispecific masked antigen binding protein binds to Epidermal Growth Factor Receptor (EGFR). The bispecific masked antigen binding proteins disclosed herein optionally bind to human EGFR with a binding constant (Kd) of ≦ 1 μ M, e.g., in some embodiments, 100nM ≦ 10nM, or ≦ 1 nM.

In preferred embodiments, the masked bispecific or multispecific antigen-binding protein binds both CD3 and EGFR.

In some embodiments, the masked antigen binding protein is a heterodimer such that it includes an antigen binding domain that is a Fab (e.g., an IgG Fab) and an antigen binding domain that is an scFv. For example, in exemplary embodiments, the masked antigen binding protein comprises heavy and light chains (e.g., IgG heavy and light chains) that bind to one target (e.g., EGFR) and an scFv domain that binds to a second target (e.g., a T cell surface antigen such as CD 3). Single chain antibodies (scfvs) are antigen binding proteins in which the VL and VH regions are joined by a linker (e.g., a synthetic sequence of amino acid residues typically about 15 to about 20 amino acids in length) to form a continuous protein chain, where the linker is sufficiently long to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al, 1988, Science [ Science ]242:423-26 and Huston et al, 1988, proc.natl.acad.sci. [ national academy of sciences ] USA 85: 5879-83). An example of a linker suitable for scFv is GGGGSGGGGSGGGS (SEQ ID NO: 199). Other exemplary linkers contain at least 4-5 amino acids, ranging from about 4 residues to about 20 residues.

An exemplary form of a masked antigen-binding protein comprises (i) two heavy chains comprising a scFv operably attached near the N-terminus of one or both heavy chains, and (ii) two light chains associated with the heavy chains to form an antigen-binding domain, wherein the scFv binds CD3 and the Fab portion binds EGFR. In this example, the antigen binding protein comprises three (or four) antigen binding domains, which typically bind two or more different antigens (or epitopes). An example of a linker suitable for linking the scFv to the heavy chain variable region is GGGGS (SEQ ID NO: 197). An exemplary linker contains at least 1 suitable linker having 4-5 amino acids.

A masked antigen binding protein comprises a Masking Domain (MD) coupled (e.g., by a covalent bond or another form of attachment) to an Antibody (AB). The Masking Domain (MD) comprises a masking peptide (or Masking Polypeptide) (MP) and a protein recognition site (PR). The masking peptide (or masking polypeptide) may be a stretch of amino acids that prevents the binding of the antigen binding domain to its antigen. Typically, masking peptides of short sequences of 5-15 amino acids in length are employed, but shorter and longer sequences (i.e., masking polypeptides) are also contemplated. Masking domains are further described, for example, in international publication No. Wo 2017/040344; U.S. patent publication No. 2015/0079088 (incorporated by reference in its entirety, and specifically for the disclosure of masking domains used with antibodies that bind EGFR) and U.S. patent publication No. 2016/0194399 (incorporated by reference in its entirety, and specifically for the disclosure of masking domains used with antibodies that bind CD 3).

In some embodiments, the masking peptide (or Masking Polypeptide) (MP) is attached to the antigen binding domain (AB) through a protein recognition site (PR), which is optionally part of a larger linker sequence (i.e., a stretch of amino acids that links the MP to the antigen binding protein). PR function as a substrate (or binding site) for proteins or proteases, preferably extracellular proteases. PR may be selected based on a protein or protease produced by cells proximal to cells expressing the target (including, e.g., tumor cells) and/or by cells co-localized in the tissue (including, e.g., tumor cells) with the desired target of the at least one AB of the masked antigen binding protein. In some embodiments, the protease is a u-type plasminogen activator (uPA, also known as urokinase), legumain, and/or a proteolytic enzyme (also known as MT-SP1 or MTSP 1). In some embodiments, the protease is a Matrix Metalloproteinase (MMP). In some embodiments, the protease is one of the proteases described in Rawlings, N and Salvesen, G.handbook of Proteolytic Enzymes (third edition). Esseville, Inc. (Elsevier),2013.ISBN: 978-0-12-382219-2. Alternatively, the MP is coupled to the antigen binding protein via a non-cleavable protein binding domain. In this regard, PR is optionally an amino acid sequence that upon interaction with, for example, a protein or protease, changes conformation such that the position of MP is adjusted and AB is free to bind to the target.

In aspects of the disclosure, a bispecific masked antigen binding protein comprises the MD of each antigen binding portion (e.g., AB1, AB2, AB3, etc.) of the construct. For example, a bispecific masked antigen binding protein comprising two Fab portions and two scfvs may comprise two sets of MDs, which may independently be the same or different. To illustrate, the bispecific masked antigen binding protein comprises (i) two scfvs that bind CD3 and MD1 attached to each scFv (optionally the same MD for each scFv), and (ii) two Fab portions that bind EGFR and MD2 attached to each Fab (optionally the same MD for each Fab). In various aspects, the PR of MD1 and MD2 comprise the same protein recognition sequence, allowing release of the MD of each binding region in the same environment upon engagement with a protease. MD can be attached at the N-terminus or C-terminus of the antigen binding domain, as long as MD is able to interfere with the binding of the antigen binding domain to the target and does not interfere with the binding once the MD linker is released. When the antigen binding domain is a Fab, the MD may be operably linked to a heavy chain variable region or a light chain variable region. Where the bispecific masked antigen binding protein comprises a whole antibody (i.e., a "stacked" conformation) having both a Fab antigen binding domain and a scFv fused to a heavy chain, the MD associated with the Fab antigen binding domain is preferably fused to a light chain variable region.

In some embodiments, one AB domain (e.g., AB1) in the bispecific masked antigen binding protein is conjugated to a T cell surface ligand (e.g., CD3), and optionally takes the form of an scFv. Exemplary masked antigen binding proteins conjugated to CD3 include, but are not limited to, international publication No. WO 2017/040344, U.S. patent publication No. 20160194399.

In some embodiments, one AB domain (e.g., AB2) in the antigen binding protein is conjugated to a tumor antigen (e.g., EGFR). Exemplary masked antigen binding proteins that bind to EGFR include, but are not limited to, those antibodies disclosed in U.S. patent publication No. 20150079088.

In some embodiments, the masked antigen binding protein (including, e.g., a bispecific masked antigen binding protein) is modified to alter the isoelectric point of the antibody. For example, in some embodiments, one or more negatively charged pH sensitive amino acids (e.g., aspartic acid or glutamic acid) in one or both masking domains are substituted with a positively charged amino acid (e.g., lysine or arginine). In some embodiments, replacing aspartic acid in one or more masking portions of an antibody can increase the pI. In some embodiments, one or more negatively charged pH sensitive amino acids (e.g., aspartic acid or glutamic acid) in one or both masking domains are removed or substituted with neutral amino acids.

While the masked antigen binding proteins of the present disclosure (including, e.g., bispecific or multispecific masked antigen binding proteins) are generally described herein as comprising an intact antibody structure, the present disclosure also contemplates the use of antigen binding antibody fragments that lack at least a portion of the traditional two heavy chain/two light chain structures. Fragments of the antigen binding protein used as a mask comprise an antigen binding domain (AB) operably linked to a Masking Domain (MD) as described above. For example, in some embodiments, the antigen binding protein comprises two scfvs linked by a suitable linker (e.g., a segment of amino acids of sufficient length to allow each scFv to bind its target). One or two scfvs are attached to the MD; when two scfvs are attached to MD, MD may be the same or different (i.e., preferably PR is the same, although MP and/or PR are different in various aspects).

The masked bispecific antibodies disclosed herein can be prepared by methods known in the art, for example, as described in international publication nos. WO 2017/040344; U.S. patent publication numbers 2015/0079088; and the method described in U.S. patent publication No. 2016/0194399.

Table 3 lists the sequences disclosed herein.

TABLE 3

The invention will be more fully understood by reference to the following examples. However, these examples should not be construed as limiting the scope of the invention.

Examples of the invention

Example 1: upon storage under refrigerated conditions (-20 ℃), the aggregation of the BiTE molecules increases

Compositions containing 1mg/mL each of HLE BiTE (MSLNxCD3, CD19xCD3, CD33xCD3, BCMAxCD3, and DLL3xCD3) and having a pH of pH 4.2 were filled into 5mL vials and stored at-20 ℃ for one month. One month later, the composition was thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates (e.g., bispecific antibody dimers) in the composition. SE-UHPLC uses a size exclusion ultra high performance analytical column to separate proteins in solution based on their hydrodynamic volume. The high molecular weight (aggregate peak) elutes earlier than the monomer and lower molecular weight peaks. The fractions were eluted isocratically, detected by UV detection, integrated, and the results reported as relative peak area percentages of the high molecular weight, main peak and low molecular weight peaks. As shown in fig. 1, the level of HMW aggregates increased in each composition after storage.

Example 2: relationship between BiTE molecular aggregation and pH and temperature

The composition comprising DLL3xCD3 HLE BiTE at a concentration of 1mg/mL and a pH of 4.2, 4.8 or 6.3 was filled into 5mL vials and stored at-20 ℃ for 1 month. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates and the results are shown in figure 2A. HMW aggregates formed during storage appeared to be pH dependent (fig. 2A).

Also, a composition comprising DLL3xCD3 HLE BiTE at a concentration of 1mg/mL was charged into a 5mL vial and stored at-10 ℃, -20 ℃, -30 ℃, -40 ℃ and-70 ℃ for one month. After one month, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates and the results are shown in fig. 2B. The level of HMW aggregates increased when the BiTE was stored at-10 ℃, -20 ℃, -30 ℃, and little increase in HMW aggregate level was observed when the BiTE was stored at temperatures below the Tg' (-32 ℃) of the composition comprising the BiTE (FIG. 2B).

Example 3: maintaining the thawed BiTE at a temperature between 15 ℃ and 30 ℃ reduces the level of aggregation

Compositions containing DLL3xCD3 HLE BiTE at concentrations of 1mg/mL, 5mg/mL and 13mg/mL were filled into 5mL vials and stored at-20 ℃ for 12 months. After 12 months, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. After the retention period, HMW aggregate levels decreased to below 5% (fig. 3A).

In a second series of experiments, compositions comprising EGFRvIIIxCD3 BiTE at concentrations of 0.5mg/mL and 2mg/mL were filled into 5mL vials and stored at-20 ℃ for one month. One month later, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. After the retention period, the HMW aggregate level decreased to below 5% (fig. 3B).

In a third series of experiments, a composition containing each of HLE BiTE (MSLNxCD3, CD19xCD3, CD33xCD3, CDH19xCD3, BCMAxCD3, DLL3xCD3, FLT3xCD3, PSMAxCD3, and CD70xCD3) at a concentration of 1mg/mL was filled into 5mL vials and stored at-20 ℃ for one month. One month later, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. After the retention period, the level of HMW aggregates in each composition decreased to below 1% (fig. 3C).

In a fourth series of experiments, a composition containing each of HLE BiTE (CD33xCD3 and DLL3xCD3) at a concentration of 1mg/mL was charged to 5mL and stored at-30 ℃ for 18 months. After 18 months, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates. The thawed compositions were kept at room temperature for an additional 24 hours and analyzed again for HMW aggregate levels using SE-UHPLC. After the retention period, the level of HMW aggregates in each composition decreased to below 0.4% (fig. 3D).

Example 4: maintaining the thawed BiTE at a temperature between 15 ℃ and 30 ℃ does not affect the stability properties of the BiTE

The composition comprising DLL3xCD3 HLE BiTE at a concentration of 13mg/mL was filled into 5mL vials and stored at-20 ℃ for 12 months. After 12 months, the composition was thawed at room temperature and the thawed composition was held at room temperature for an additional 24 hours. After a 24 hour holding period, several stability attributes were analyzed and the results are shown in table 4.

The potency of HLE BiTE molecules was measured by a cell-based bioassay that measures cell death by loss of luminescence in cancer cell lines. By comparing the test sample response to the response of the reference standard (relative potency), the biological activity of the test sample can be determined.

The shear of the BiTE molecules was measured using reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS). rCE-SDS separates proteins based on hydrodynamic size under reducing and denaturing conditions. Proteins were denatured, reduced, and injected into bare fused silica capillaries filled with a polymer gel matrix. Applying a voltage across the capillary and separating the SDS-coated proteins based on their hydrodynamic size; smaller size proteins migrate earlier than larger size proteins. Proteins were detected using a photodiode array (PDA) detector, integrated, and the results were reported as relative peak area percentages of low molecular weight, main peak, and high molecular weight peaks.

Charge variants of BiTE molecules were measured using cation exchange high performance liquid chromatography (CEX-HPLC). At the appropriate pH, the charge variants of the protein are eluted using a mobile phase gradient of increasing ionic strength. Proteins with less positive surface charge elute earlier than proteins with more positive surface charge. Eluted charge variants were detected by UV detection, integrated and the results of the main, acidic and basic peaks were reported as a percentage of the total peak area.

As shown in table 4, BiTE remaining thawed had no negative effect on stability attributes, while the level of HMW decreased after the retention period.

TABLE 4

HMW-high molecular weight species, RP-relative potency, MP-main peak, LMW-low molecular weight species

Example 5: reduction of HMW levels of BiTE molecules after storage under freezing conditions as a function of retention time and retention temperature

Compositions comprising 1mg/mL HLE BiTE (CD33xCD3 and DLL3xCD3) and 2mg/mL BiTE (EGFRvIIIxCD3) each were filled into 5mL vials and stored at-20 ℃ for one month. One month later, the composition was thawed at room temperature and immediately analyzed by size exclusion ultra high performance liquid chromatography (SE-UHPLC) to determine the level of HMW aggregates (e.g., bispecific antibody dimers) in the composition.

The thawed compositions were held at various temperatures for a maximum hold time of up to 96 hours and analyzed for HMW aggregate levels by SE-UHPLC at various time points. The retention time required for HMW aggregate levels to decrease to the initial pre-freezing levels of CD33xCD3 and DLL3xCD3 HLE BiTE and egfrviii xCD3 BiTE at various temperatures was the same and is shown in figure 4.

Example 6: stabilization of Benzyl Alcohol (BA).

Compositions containing 1mg/mL each of HLE BiTE (CD19xCD3, CD33xCD3, BCMAxCD3, DLL3xCD3, and EGFRvIIIxCD3) were charged to 5mL and stored at-20 ℃ for four weeks in the presence and absence of BA. After four weeks, the compositions were thawed at room temperature and immediately analyzed by SE-UHPLC to determine the level of HMW aggregates. The presence of BA stabilized BiTE during storage (fig. 5).

All references cited in this application are incorporated herein by reference

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